SID nucleic acids and polypeptides selected from a pathogenic strain of hepatitis C virus and applications thereof

ABSTRACT

The present invention relates to nucleic acids encoding SID® polypeptides which bind selectively to a polypeptide encoded by a pathogenic strain of the hepatitis C virus, as well as to the SID® polypeptides which are encoded by said nucleic acids.  
     The invention also concerns vectors comprising a nucleic acid encoding a SID® polypeptide as well as host cells transformed with such vectors.  
     The invention is also directed to two-hybrid methods which make use of the nucleic acids encoding a SID® polypeptide selected from a pathogenic strain of the hepatitis C virus as well as to methods for selecting molecules which inhibit the binding between a SID® polypeptide and a polypeptide which specifically binds thereto.

FIELD OF THE INVENTION

[0001] The present invention relates to nucleic acids encoding SID® polypeptides which bind selectively to a polypeptide encoded by a pathogenic strain of the hepatitis C virus, as well as to the SID® polypeptides which are encoded by said nucleic acids.

[0002] The invention also concerns vectors comprising a nucleic acid encoding a SID® polypeptide as well as host cells transformed with such vectors.

[0003] The invention is also directed to two-hybrid methods which make use of the nucleic acids encoding a SID® polypeptide selected from a pathogenic strain of the hepatitis C virus as well as to methods for selecting molecules which inhibit the binding between a SID® polypeptide and a polypeptide which specifically binds thereto.

[0004] The invention also pertains to marker compounds containing a SID® polypeptide as well as nucleic acids encoding such marker compounds and methods and kits using the same.

BACKGROUND OF THE INVENTION

[0005] The hepatitis C virus (HCV) causes several liver diseases, including liver cancer. The HCV genome is a plus-stranded RNA that encodes the single polyprotein processed into at least 10 mature polypeptides.

[0006] The structural proteins are located in the amino terminal quarter of the polyprotein, and the non-structural (NS) polypeptides in the remainder (for a review, see HOUGHTON, 1996). The genome organisation resembles that of flaviviruses and pestiviruses and HCV is now considered to be a member of the flaviviridae family.

[0007] The gene products of HCV are, from the N-terminus to the C-terminus: core (p22), E1 (gp35), E2 (gp70), NS2(p21), NS3 (p70), NS4a (p4), NS4b(p27), NS5a (p58), NS5b (p66), as disclosed in FIG. 1. Core, E1and E2 are the structural proteins of the virus processed by the host signal peptidase(s). The core protein and the genomic RNA constitute the internal viral core and E1 and E2 together with lipid membrane constitute the viral envelop (DUBUISSON et al., 1994; GRAKOUI et al., 1993; HIGIKATA et al., 1993.).

[0008] The NS proteins are processed by the viral protein NS3 which has two functional domains: one (Cro-1), encompassing the NS2 region and the N-terminal portion of NS3, which cleaves autocatalytically between NS2 and NS3, and the other (Cro-2), located solely in the N-terminal portion of NS3, cleaves the other sites downstream NS3 (BARTENSCHLAGER et al; 1995; HIGIKATA et al;, 1993).

[0009] Various HCV protein-protein interactions have already been identified, notably by two hybrid methods. Noticeably, FLAJOLET et al; (2000) have shown interactions between NS3 and NS4A proteins as well as between NS4A and NS2 proteins. These authors have also shown core-core, NS3-E2, NS5A-E1, NS4A-NS3 and NS4A-NS2 interactions. Covalent as well as non-covalent interactions between E1 and E2 have been shown by PATEL et al; (1999). The protein interactions between NS3 and the HCV RNA helicase have also been described (MIN et al; 1999; GALLINARI et al., 1999) as well as interaction between NS3 and NS4A (URBANI et al., 1999; DI MARCO et al., 2000; BUTKIEWICZ et al., 2000).

[0010] However, the prior art methods allow the determination of interactions between full length proteins or large domains of proteins encoded by the genome of the hepatitis C virus which may contain more than one region of interaction with one or several HCV proteins. BUTKIEWICZ et al. (2000) discloses the interaction between the NS3 protease and a small peptide derived from NS4A. However, BUTKIEWICZ et al. (2000) discloses exclusively in vitro assays for interactions between the small peptides derived from NS4A and the NS3 protease from HCV which may not be of physiological relevance.

[0011] There is a need in the art for polypeptides that contain the minimal aminoacid sequence that is able to bind specifically with a naturally-occurring HCV protein in physiological conditions in order to design new tools for therapeutic and detection purposes related to HCV.

SUMMARY OF THE INVENTION

[0012] This invention provides nucleic acids encoding polypeptides, which are termed SID® polypeptides, wherein these polypeptides are the final products of a double selection method involving a first step of selection of HCV-derived polynucleotides through a two-hybrid system and a second selection step involving an alignment between the different polynucleotides selected at the first step.

[0013] The invention also pertains to the SID® polypeptides encoded by the SID® nucleic acids.

[0014] Another object of the invention are recombinant vectors containing a SID® nucleic acid as defined above as well as host cells transformed with such vectors or nucleic acids.

[0015] A further object of the invention consists of two-hybrid methods which make use of these SID® nucleic acids as well as to methods for selecting molecules which inhibit the binding between a SID® polypeptide and a polypeptide that binds specifically thereto, as well as kits for performing these methods.

[0016] It is still a further object of the invention to provide for marker compounds which comprise a SID® polypeptide or which are encoded by a polynucleotide containing a SID® nucleic acid as defined above, as well as to methods and kits which make use of these marker compounds.

[0017] This invention also relates to pharmaceutical compositions as well as to methods for preventing or curing a HCV viral infection in a human or an animal that use a SID® polypeptide or a SID® nucleic acid as disclosed herein.

[0018] Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specifications, referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 consists of a general overview of HCV genome and its encoded polyprotein. The RNA coding strand is represented with a line for untranslated regions (NCR) and boxes for coding regions.

[0020] Positions and enzymes responsible for cleavage are indicated above. p7 is a secondary cleavage product of E2 (adapted from HOUGHTON, 1996).

[0021]FIG. 2 is a restriction map of the plasmid pAS2ΔΔ which may be used for producing a recombinant “Selected Interacting Domain (SID®)” polypeptide or a recombinant marker compound of the invention.

[0022]FIG. 3 is a restriction map of the plasmid pACTII which may be used for producing a recombinant “Selected Interacting Domain (SID®)”.

[0023]FIG. 4 is a restriction map of the plasmid pUT18 which may be used for producing a recombinant “Selected Interacting Domain (SID®)”.

[0024]FIG. 5 is a restriction map of the plasmid pUT18C which may be used for producing a recombinant “Selected Interacting Domain (SID®)”.

[0025]FIG. 6 is a restriction map of the plasmid pT25 which may be used for producing a recombinant “Selected Interacting Domain (SID®)”.

[0026]FIG. 7 is a restriction map of the plasmid pKT25 which may be used for producing a recombinant “Selected Interacting Domain (SID®)”.

[0027]FIG. 8 is an illustration of the first step of selecting a SID® nucleic acid of the invention, wherein it is performed a selection of different sets of overlapping nucleic acids primarily selected through a two-hybrid method, in order to define pre-SID nucleic acids. Three fragments frg1, frg2 and frg3 of lengths l1, l2 and l3 respectively. Fragment l1 and l2 are clustered together if the length of intersection, I, is greater than 30% of l1 and l2. Fragment frg3 is grouped with fragments frg and frg2 if the length of intersection between frg1 and frg3, l′, is greater than 30% of l1 and l3 and if the length of intersection between frg 2 and frg 3, l>>, is greater than 30% of l2 and l3.

[0028]FIG. 9 illustrates the selection of pre-SID® nucleic acid from a particular set of overlapping nucleic acids previously selected through a two-hybrid method. The pre-SID® is defined as the intersection of all the fragments (frg1-6) in a cluster.

[0029]FIG. 10 illustrates the selection of a SID® nucleic acid from the overlapping regions between two pre-SID nucleic acids. A SID® is defined if the length of overlap between two pre-SID®s, l, is greater than 30 bp. Further SID®s are defined by non-overlapping areas if their length (l′) represents more than 30% of the length of one of the fragments which contributes to the corresponding pre-SID® (frg1-6).

[0030]FIG. 11 illustrates a further step of determining SID® nucleic acids after alignment of two overlapping SID nucleic acids identified according to FIG. 10. Fragments frg1′ and frg2′ contribute to both SID®1 and SID®2 (top panel). For each SID®, the number of fragments are counted and fragments are assigned to the SID® with the most fragments. The remaining fragments are re-analysed and a new SID® is defined as the region of intersection of these fragments (bottom panel, SID®2′—fragment 3′ and fragment 4′.

[0031]FIG. 12 illustrates a map of the vector pB5 which may be used in example 1.

[0032]FIG. 13 illustrates a map of the vector pP6 which may be used in example 1.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention firstly provides for nucleic acids encoding SID® polypeptides.

[0034] As generally used herein, a <<bait>> nucleic acid encodes a <<bait>> polypeptide. A polypeptide is termed a <<bait>> polypeptide when this polypeptide is used to select a formerly unknown <<prey>> nucleic acid encoding a <<prey>> polypeptide which binds selectively with said <<bait>> polypeptide. Indeed, a <<prey>> nucleic acid which has been selected for binding to a given bait polypeptide may be used in another selection method or in another round of the same selection method as a <<bait>> nucleic acid encoding a <<bait>> polypeptide for the purpose of selection of new prey nucleic acids, encoding prey polypeptides which bind selectively with said bait polypeptide, it being understood that the nucleic acid encoding said bait polypeptide was formerly selected from a population of prey nucleic acids.

Selected Interacting Domain (SID®) Polypeptides and Methods for their Preparation

[0035] A selected interacting domain polypeptide that binds specifically to a polypeptide of interest is the result of a two-step screening procedure, wherein:

[0036] 1) the first step consists of selecting and characterizing a collection of nucleic acids (prey nucleic acids) encoding polypeptides which bind specifically to a given bait polypeptide of interest; and

[0037] 2) the second step of the two-step procedure consists of determining the nucleic acid sequences which encode for SID® polypeptides after having generated sets of polynucleotides from the collection of nucleic acids selected at step 1).

[0038] As a result of the original two-step screening procedure disclosed hereunder, every nucleic acid finally selected encodes a <<Selected Interacting Domain (SID®)” polypeptide which binds with a high specificity with the bait polypeptide of interest.

[0039] Step 1) Selecting Prey Nucleic Acids

[0040] The first step of selecting a collection of nucleic acids encoding polypeptides which binds specifically to the bait polypeptide is carried out through a yeast two-hybrid system. The yeast two-hybrid system is designed to study protein-protein interactions in vivo, and relies upon the fusion of a bait protein to the DNA binding domain of the yeast Gal4 protein.

[0041] According to the present invention, the first step of the procedure for selecting a Selected Interacting Domain (SID®) polynucleotide encoding a Selected Interacting Domain (SID®) polypeptide consists of the two-hybrid screening system described by Fromont-Racine et al. (1997) or the method described by FLAJOLET et al. (2000). The yeast two-hybrid system utilizes hybrid proteins to detect protein-protein interactions by means of direct activation of a reporter gene expression. In essence, the nucleic acids encoding the two putative protein partners, the bait polypeptide of interest and the prey polypeptide, are genetically fused to the DNA-binding domain of a transcription factor and to a transcriptional activation domain, respectively.

[0042] Construction of the Prey HCV Nucleic Acids Library

[0043] Then, a genomic DNA library prepared from the genome of the pathogenic H77 strain of HCV (Yanagi et al., 1997), is constructed in the specially designed vector pP6 shown in FIG. 13 after ligation to suitable linkers, such that every genomic DNA insert is fused to a nucleotide sequence in the vector that encodes the transcription of domain of the Gal4 protein.

[0044] The polypeptides encoded by the nucleotide inserts of the genomic DNA library thus prepared are termed “prey” polypeptides in the context of the presently described selection method of prey nucleic acids.

[0045] Construction of the Bait Nucleic Acids Library

[0046] The DNA fragments obtained after nebulization of the HCV genomic DNA are also inserted in plasmid pB5 shown in FIG. 12 wherein these DNA inserts are fused to a polynucleotide encoding the DNA binding domain of the Gal4 protein and the recombinant vectors are used to transform E. coli cells. The transformed E. coli cells are grown and plasmid DNA is extracted and sequenced.

[0047] These plasmids which code in frame fusion proteins are used as bait plasmids. Bait plasmids thus consist of a collection of recombinant pB5 plasmids each containing inserted therein a DNA fragment from the H77 strain HCV genome encoding a polypeptide consisting of all or part of a HCV protein or alternatively a polypeptide consisting of all or part of two HCV proteins encoded by contiguous nucleic acid sequences of the HCV genome.

[0048] The selected HCV bait nucleic acids of the invention are referred to as the nucleotide sequences SEQ ID N^(o)114 to 150.

[0049] The selected HCV bait polypeptides encoded by the nucleic sequences SEQ ID N^(o)114 to 150 consist respectively of the aminoacid sequences SEQ ID N^(o)77 to 113.

[0050] Detectable marker genes are already present within the chromosomic yeast DNA and consist respectively of the His3 and LacZ genes, such as described by FROMONT-RACINE et al. (1997) or FLAJOLET et al. (2000).

[0051] Then, the collection of nucleic acid inserts contained in the collection of E. Coli cell clones containing the genomic DNA or HCV DNA library previously prepared are used to transform a first yeast strain, namely the Y187 Saccharomyces cerevisiae strain (phenotype:MATα, Gal4Δ, gal80Δ, ade2-101, His3, Leu2-3, −112 Trp1-901, Ura3-52, URA3::UASGAL1-LacZ Met).

[0052] The nucleic acid encoding the bait polypeptide of interest is inserted in the appropriate vector, said vector being used to transform a second yeast strain which may be the CG1945 (MATa Gal4-542 Gal180-538, Ade2-101, His3*200, Leu2-3, −112 Trp1-901 Ura3-52, Lys2-801, URA3::GAL4 17Mers (X3)-CyC1TATA-LacZ LYS2::GAL1 UAS-GAL1TATA-His3 CYH^(R)).

[0053] Then, the two yeast strains are mated to obtain a collection of mated cells.

[0054] The clones derived from the collection of mated cells above which are positive in an X-Gal overlay assay are those for which an interaction between the recombinant bait polypeptide and a polypeptide encoded by a nucleic acid insert originating from the HCV genomic library has occurred.

[0055] The clones derived from the collection of mated cells above may also be selected in the presence of histidine, and the positive clones are those for which an interaction between the recombinant bait polypeptide and a polypeptide encoded by a nucleic acid insert originating from the HCV genomic library has occurred.

[0056] In a further step, the prey nucleic acid inserts contained in the positively selected clones are amplified and sequenced.

[0057] Step 2: Determination of the Nucleic Acid Sequences Encoding a Selected Interacting Domain (SID®) Polypeptide which Binds Specifically to a Bait Polypeptide of Interest

[0058] This is the second step of the two step procedure defined above, which allows the precise selection of nucleic acids encoding the SID® nucleic acids of the present invention which are derived from the H77 strain HCV genome.

[0059] The SID® nucleic acid selection procedure, which is disclosed hereunder, has been specifically designed for the HCV genome which encodes for a single polyprotein and which thus comprises contiguous Open Reading Frames, said polyprotein being further processed to produce at least 10 mature structural and non-structural viral proteins.

[0060] Thus, the second selection step of the two-step procedure consists of a method for determining a polynucleotide encoding a Selected Interacting Domain (SID®) of a prey polypeptide of interest derived from HCV, which prey polypeptide interacts with a bait polypeptide, wherein said method comprises the steps of:

[0061] a) selecting, from the collection of prey polynucleotides obtained at the end of the first step of the two-step procedure described herein, all prey polynucleotides encoding a prey polypeptide capable of interacting with said bait polypeptide and containing a common nucleic acid fragment;

[0062] b) aligning the nucleotide sequences of the prey polynucleotides selected at step a) and gathering in one set or in a plurality of sets of sequences those nucleotide sequences which have sequences that overlap for more than 30% of their respective nucleic acid length, wherein each common overlapping nucleotide sequence in one set of sequences defines a sequence encoding a pre-SID® polypeptide (see FIGS. 8 and 9); and

[0063] c) aligning two sequences encoding two respective pre-SID® polypeptides (see FIG. 10), and:

[0064] i) defining an overlapping nucleic acid sequence between the sequences encoding the two respective pre-SID® polypeptides as a sequence encoding a SID® polypeptide, provided that the overlapping sequence is of at least 30 nucleotides in length;

[0065] ii) defining a non-overlapping nucleic acid sequence between the sequences encoding the two respective pre-SID® polypeptides as a sequence encoding a SID® polypeptide, provided that (1) said non-overlapping sequence has more than 30 nucleotides in length and (2) said non-overlapping sequence represents at least 30% in length of any one of the polynucleotides contained in the set of prey polynucleotides used for defining the sequence encoding each pre-SID® polypeptide.

[0066] This method may further comprise the steps of:

[0067] d) counting the number of overlapping prey polynucleotides contained in a first set of polynucleotides defining a sequence encoding a first SID® polypeptide;

[0068] e) counting the number of overlapping prey polynucleotides contained in a second set of polynucleotides defining a sequence encoding a second SID® polypeptide which overlaps with the sequence encoding the first SID® polypeptide;

[0069] f) determining which sequence among those encoding respectively the first SID® polypeptide and the second SID® polypeptide has been defined with the largest number of prey polynucleotides and selecting this set of prey sequences.

[0070] g) adding to the set of prey sequences selected at step f) those sequences that were contained in the set of prey sequences used for defining the sequence encoding the SID® polypeptide with the smallest number of prey sequences and which overlap with the sequence encoding the SID® polypeptide with the largest number of prey sequences.;

[0071] h) aligning the prey sequences added at step g) with the sequences already contained in the set of prey sequences which defined the sequence encoding the SID® polypeptide with the largest number of prey sequences;

[0072] i) defining an overlapping sequence between the whole sequences which were aligned in step h), wherein said overlapping sequence consists of a sequence encoding a SID® polypeptide. (See FIG. 11).

[0073] The method for selecting a SID® nucleic acid encoding a SID® polypeptide is an object of the present invention, as well as any SID® nucleic acid or any SID® polypeptide which may be obtained by this selection method.

[0074] SID® Nucleic Acids of the Invention

[0075] The SID® nucleic acids selected as described above starting from the genome of the H77 strain of HCV are the nucleic acid sequences of SEQ ID N^(o)39 to 76 which encode the SID® polypeptides of SEQ ID N^(o)1 to 38.

[0076] A first object of the invention consists of a nucleic acid which encodes a polypeptide selected from the group consisting of the aminoacid sequences SEQ ID N^(o)1 to 38 or a variant thereof, and a sequence complementary thereto.

[0077] For the purposes of the present invention, a first polynucleotide is considered as being <<complementary>> to a second polynucleotide when each base of the first polynucleotide is paired with the complementary base of the second polynucleotide whose orientation is reversed. The complementary bases are A and T(or A and U), or C and G.

[0078] Preferably, any one of the nucleic acid or the polypeptides encompassed by the invention is under a purified or an isolated form.

[0079] The term “isolated” for the purposes of the present invention designates a biological material (nucleic acid or protein) which has been removed from its original environment (the environment in which it is naturally present).

[0080] For example, a polynucleotide present in the natural state in a plant or an animal is not isolated. The same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted in the genome of the plant or animal is considered as being “isolated”.

[0081] Such a polynucleotide may be included in a vector and/or such a polynucleotide may be included in a composition and remains nevertheless in the isolated state because of the fact that the vector or the composition does not constitute its natural environment.

[0082] The term “purified” does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. It is rather a relative definition.

[0083] A polynucleotide is in the “purified” state after purification of the starting material or of the natural material by at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

[0084] “Isolated polypeptide” or “isolated protein” is a polypeptide or protein which is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilisers, or compounding into a pharmaceutically acceptable preparation.

[0085] Variants of a Selected Interacting Domain (SID®) Polypeptide and Nucleic Acids Encoding them

[0086] As intended herein, a variant of a Selected Interacting Domain (SID®) polypeptide may be either a variant polypeptide of the Selected Interacting Domain (SID®) polypeptide or a polypeptide which is encoded by a nucleic acid variant of the polynucleotide encoding said Selected Interacting Domain (SID®) polypeptide.

[0087] Polynucleotides which encode a polypeptide variant of a Selected Interacting Domain (SID®) polypeptide, as the term is used herein, are polynucleotides that differ from the reference polynucleotide encoding the parent SID® polypeptide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the reference polynucleotide may be generated by mutagenesis techniques, including those applied to polynucleotides, cells or organisms well known to one skilled in the art.

[0088] Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.

[0089] Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95% identical after optimal alignment to the reference polynucleotide of SEQ ID N^(o)39 to 76 encoding the reference Selected Interacting Domain (SID®) polypeptide, preferably at least 96%, 97%, 98% and most preferably at least 99% identical to the reference polynucleotide. Similarly, a variant of a SID® polypeptide of the invention consists of a polypeptide having at least 95% aminoacid identity with a polypeptide selected from the aminoacid sequences SEQ ID N^(o)1 to 38, and preferably at least 96%, 97%, 98% and most preferably at least 99% aminoacid identity with one of SEQ ID N^(o)1 to 38.

[0090] Identity refers to sequence identity between two peptides or between two nucleic acid molecules. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical aminoacids at positions shared by these sequences. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1972), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Solftware Package Release 7.0, Genetics Computer Group, 575, Science Dr. Madison, W1), or by inspection. The best alignment (i.e., resulting in the highest percentage of identity over the comparison window) generated by the various methods is selected. The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, U or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0091] Most preferably, the percentage of nucleic acid or aminoacid identity between two nucleic acid or aminoacid sequences is calculated using the BLAST software (Version 2.06 of September 1998) with the default parameters.

[0092] Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the aminoacid encoded by the reference polynucleotide.

[0093] However, nucleotide changes may also result in aminoacid substitutions, additions, deletions, fusions and truncations in the Selected Interacting Domain (SD®) polypeptide encoded by the reference sequence.

[0094] The substitutions, deletions or additions may involve one or more nucleotides. Alterations may produce conservative or non-conservative aminoacid substitutions, deletions or additions.

[0095] Most preferably, the variant of a Selected Interacting Domain (SID®) polypeptide encoded by a variant polynucleotide possesses at least the same affinity of binding to its protein or polypeptide counterpart, against which it has been initially selected as described above.

[0096] The affinity of a given SID® polypeptide of the invention for a polypeptide into which it specifically binds is defined as the affinity constant Ka, wherein ${Ka} = \frac{\left\lbrack {{{SID®}/{polypeptide}}\quad {complex}} \right\rbrack}{\left\lbrack {{free}\quad {SID®}} \right\rbrack \left\lbrack {{free}\quad {polypeptide}} \right\rbrack}$

[0097] with [free SID®], [free polypeptide] and [SID®/polypeptide complex] consist of the concentrations at equilibrium respectively of the free SID® polypeptide, of the free polypeptide onto which the SID® polypeptide specifically binds and of the complex formed between the SID® polypeptide and the polypeptide onto which said SID® polypeptide specifically binds.

[0098] Most preferably, the affinity of a SID® polypeptide of the invention or a variant thereof for its polypeptide counterpart (polypeptide partner) is assessed on a Biacore™ apparatus marketed by Amercham Pharmacia Biotech Company such as described by SZABO et al. (1995) and by Edwards and Leartherbarrow (1997).

[0099] As used herein, the expression <<at least the same affinity>> with reference to the affinity of binding between a SID® polypeptide of the invention to another polypeptide means that the Ka is identical or is of at least two-fold, preferably at least three-fold and most preferably at least five-fold greater than the Ka value of reference.

[0100] In another preferred embodiment, the variant of a Selected Interacting Domain (SID®) polypeptide which is encoded by a variant polynucleotide of the invention possesses a higher specificity of binding to its counterpart polypeptide or protein than the reference Selected Interacting Domain (SID®) polypeptide.

[0101] A variant of a Selected Interacting Domain (SID®) polypeptide according to the invention may be (1) one in which one or more, most preferably from one to three, of the aminoacid residues are substituted with a conserved or a non-conserved aminoacid residue and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (2) one in which one or more of the aminoacid residues includes a substituent group.

[0102] In the case of an aminoacid substitution in the aminoacid sequence of a Selected Interacting Domain (SID®) polypeptide according to the invention, one or several-consecutive or non-consecutive—aminoacids are replaced by “equivalent” aminoacids. The expression “equivalent” aminoacid is used herein to designate any aminoacid that may be substituted for one of the aminoacids belonging to the native Selected Interacting Domain (SID®) polypeptide structure without decreasing the binding properties of the corresponding peptides to their counterpart polypeptide or protein, as regards the reference Selected Interacting Domain (SID®) polypeptide.

[0103] These equivalent aminoacids may be determined either by their structural homology with the initial aminoacids to be replaced, by the similarity of their net charge or of their hydrophobicity.

[0104] By an equivalent aminoacid according to the present invention is also meant the replacement of a residue in the L-form by a residue in the D-form or the replacement of a glutamic acid residue by a pyro-glutamic acid compound. The synthesis of peptides containing at least one residue in the D-form is, for example, described by KOCH (1977). A specific embodiment of a variant of a Selected Interacting Domain (SID®) polypeptide according to the invention includes, but is not limited to, a peptide molecule which is resistant to proteolysis, such as a peptide in which the —CONH— peptide bond is modified and replaced by a —CH₂NH—) reduced bond, a (—NHCO—) retroinverso bond, a (—CH₂—O—) methylene-oxy bond, a (—CH₂—S—) thiomethylene bond, a (—CH₂CH₂—) carba bond, a (—CO—CH₂) hydroxyethylene bond, a (—N—N—) bond or also a —CH═CH bond.

[0105] As used herein, a variant of a SID® polypeptide of the invention also encompasses a polypeptide having an aminoacid sequence consisting of at least:

[0106] 45 consecutive aminoacids of SEQ ID N^(o)1;

[0107] 30 consecutive aminoacids of SEQ ID N^(o)2;

[0108] 65 consecutive aminoacids of SEQ ID N^(o)3;

[0109] 30 consecutive aminoacids of SEQ ID N^(o)4;

[0110] 130 consecutive aminoacids of SEQ ID N^(o)5;

[0111] 25 consecutive aminoacids of SEQ ID N^(o)6;

[0112] 23 consecutive aminoacids of SEQ ID N^(o)7.

[0113] 48 consecutive aminoacids of SEQ ID N^(o)8;

[0114] 36 consecutive aminoacids of SEQ ID N^(o)9;

[0115] 25 consecutive aminoacids of SEQ ID N^(o)10;

[0116] 24 consecutive aminoacids of SEQ ID N^(o)11;

[0117] 37 consecutive aminoacids of SEQ ID N^(o)12;

[0118] 25 consecutive aminoacids of SEQ ID N^(o)13;

[0119] 30 consecutive aminoacids of SEQ ID N^(o)14;

[0120] 27 consecutive aminoacids of SEQ ID N^(o)15;

[0121] 69 consecutive aminoacids of SEQ ID N^(o)16;

[0122] 130 consecutive aminoacids of SEQ ID N^(o)17;

[0123] 33 consecutive aminoacids of SEQ ID N^(o)18;

[0124] 25 consecutive aminoacids of SEQ ID N^(o)19;

[0125] 40 consecutive aminoacids of SEQ ID N^(o)20;

[0126] 78 consecutive aminoacids of SEQ ID N^(o)21;

[0127] 39 consecutive aminoacids of SEQ ID N^(o)22;

[0128] 57 consecutive aminoacids of SEQ ID N^(o)23;

[0129] 26 consecutive aminoacids of SEQ ID N^(o)24;

[0130] 68 consecutive aminoacids of SEQ ID N^(o)25;

[0131] 34 consecutive aminoacids of SEQ ID N^(o)26;

[0132] 42 consecutive aminoacids of SEQ ID N^(o)27;

[0133] 48 consecutive aminoacids of SEQ ID N^(o)28.

[0134] 102 consecutive aminoacids of SEQ ID N^(o)29:

[0135] 49 consecutive aminoacids of SEQ ID N^(o)30:

[0136] 92 consecutive aminoacids of SEQ ID N^(o)31;

[0137] 49 consecutive aminoacids of SEQ ID N^(o)30;

[0138] 92 consecutive aminoacids of SEQ ID N^(o)31;

[0139] 71 consecutive aminoacids of SEQ ID N^(o)32;

[0140] 55 consecutive aminoacids of SEQ ID N^(o)33;

[0141] 69 consecutive aminoacids of SEQ ID N^(o)34;

[0142] 23 consecutive aminoacids of SEQ ID N^(o)35;

[0143] 33 consecutive aminoacids of SEQ ID N^(o)36;

[0144] 32 consecutive aminoacids of SEQ ID N^(o)37; and

[0145] 22 consecutive aminoacids of SEQ ID N^(o)38.

[0146] Without wishing to be bound by any particular theory, the inventors believe that polypeptides having an aminoacid length of about 10% lesser than the aminoacid length of anyone of the SID® polypeptides of SEQ ID N^(o)1 to 39 of the invention have a high probability to retain the binding properties to a given (bait) polypeptide of the parent SID® polypeptide.

[0147] The invention also pertains to a nucleic acid encoding a SID® polypeptide which is selected from the group consisting of the sequences SEQ ID N^(o)39 to 76, and a sequence complementary thereto.

[0148] The invention is also directed to a nucleic acid encoding a variant of SID® polypeptide selected from the group consisting of the sequences SEQ ID N^(o)39 to 76, in reference to the definition of the SID® polypeptide variants above.

[0149] For example, a nucleic acid encoding a polypeptide having an aminoacid sequence consisting of at least 45 consecutive aminoacids of SEQ ID N^(o)1 comprise at least 135 (45×3) consecutive nucleotides of the polynucleotide of SEQ ID N^(o)39.

[0150] The same definition also apply for nucleic acids encoding variants of the SID® polypeptides of SEQ ID N^(o)2 to 38, which are part of the invention.

[0151] The invention further relates to a nucleic acid encoding a polypeptide having an aminoacid sequence comprising from 1 to 3 substitutions, additions or deletions of one aminoacid as regards a polypeptide selected from the group consisting of the aminoacid sequences SEQ ID N^(o)1 to 38 or a sequence complementary thereto.

[0152] Another object of the invention consists of a polypeptide selected from the group consisting of the aminoacid sequences SEQ ID N^(o)39 to 76 or a variant thereof.

[0153] Are encompassed in the family of variants of a SID® polypeptide of the invention those polypeptides having an aminoacid sequence comprising from 1 to 3 substitutions, additions or deletions of one aminoacid as regards a polypeptide selected from the group consisting of the aminoacid sequences SEQ ID N^(o)1 to 38.

[0154] The invention is also directed to an antibody directed against a a SID® polypeptide as defined above, or to a variant thereof.

[0155] The antibodies directed specifically against the Selected Interacting Domain (SID®) polypeptide or a variant thereof may be indifferently radioactively or non-radioactively labelled.

[0156] Monoclonal antibodies directed against a SID® polypeptide may be prepared from hybridomas according to the technique described by Kohler and Milstein in 1975. Polyclonal antibodies may be prepared by immunization of a mammal, especially a mouse or a rabbit, with the SID® polypeptide that is combined with an adjuvant of immunity, and then by purifying the specific antibodies contained in the serum of the immunized animal on a affinity chromatography column on which has previously been immobilized the polypeptide that has been used as the antigen.

[0157] Antibodies directed against a SID® polypeptide may also be produced by the trioma technique and by the human B-cell hybridoma technique (Kozbor et al., 1983).

[0158] Antibodies directed to a SID® polypeptide include chimeric single chain Fv antibody fragments (U.S. Pat. No. 4,946,778; Martineau et al., 1998), antibody fragments obtained through phage display libraries (Ridder et al., 1995) and humanized antibodies (Reinmann et al., 1997; Leger et al., 1997). Also, transgenic mice, or other organisms such as other mammals, may be used to express antibodies, including for example, humanized antibodies directed against a SID® polypeptide of the invention, or a variant thereof.

Vectors of the Invention

[0159] The nucleic acids coding for a Selected Interacting Domain (SID®) polypeptide or a variant thereof, which are defined in the section above, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such transcription elements include a regulatory region and a promoter as defined previously. Thus, the nucleic acid encoding a marker compound of the invention is operably linked with a promoter in a expression vector, wherein said expression vector may include a replication origin.

[0160] The necessary transcriptional and translation of signals is most preferably provided by the recombinant expression vector.

[0161] Structure of the Vectors Encompassed by the Invention

[0162] A wide variety of host/expression vector combinations may be employed in expressing the nucleic acids of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., Escherichia coli plasmids col EI, pCR1, pBR322, pMaI-C2, pET, pGEX (Smith et al., 1988), pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage I, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

[0163] For example, in a baculovirus expression system, both non-fusion transfer vectors, such as but not limited to pVL941 (BamH1cloning site; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, BglII, and PstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI, and BamH1cloning site; Summers and Invitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning site, with blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as but not limited to pAc700(BamH1 and KpnI cloning site, in which the BamH1 recognition site begins with the initiation codon; Summers), pAc701and pAc702(same as pAc700, with different reading frames), pAc360 (BamH1 cloning site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen(195)), and pBlueBacHisA, B, C (three different reading frames, with BamH1, BglII, PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBond purification, and blue/white recombinant screening of plaques; Invitrogen (220) can be used.

[0164] Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e.g., any expression vector with a DHFR expression vector, or a DHFR/methotrexate co-amplification vector, such as pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR; Kaufman, 1991). Alternatively, a glutamine synthetase/methionine sulfoximine co-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celltech). In another embodiment, a vector that directs episomal expression under control of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible methallothionein lIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamH1 cloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminal peptide purifiable via ProBond resin and cleaved by enterokinase; Invitrogen). Selectable mammalian expression vectors for use in the invention include pRc/CMV (HindIII, BstXI, NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selection; Invitrogen), and others. Vaccinia virus mammalian expression vectors (see, Kaufman, 1991, supra) for use according to the invention include but are not limited to pSC11 (SmaI cloning site, TK- and b-gal selection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamH1, ApaI, NheI, SacII, KpnI, and HindIII cloning site; TK- and b-gal selection), and pTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamH1, and Hpa cloning site, TK or XPRT selection).

[0165] Yeast expression systems can also be used according to the invention to express a Selected Interacting Domain (SID®) polypeptide or a variant thereof and also a marker compound as defined herein. For example, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcorI, BstXI, BamH1, SacI, KpnI, and HindIII cloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotIl, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention.

[0166] Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

[0167] Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992; Wu and Wu, 1988; Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0168] A cell has been “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change.

[0169] For introducing a vector in a cell host, explicit reference is made to research carried out by the group of E. Wagner, relating to gene delivery by means of plasmid-polylysine complexes (Curiel et al., 1991; and Curiel et al., 1992). The plasmid-polylysine complex investigated upon exposition to certain cell lines showed at least some expression of the gene. Further, it was found that the expression efficiency increased considerably due to the binding of transferrin to the plasmid-polylysine complex. Transferrin gives rise to close cell-complex contact with cells comprising transferrin receptors; it binds the entire complex to the transferrin receptor of cells. Subsequently, at least part of the entire complex was found to be incorporated in the cells investigated.

[0170] Several different approaches have been developed for gene transfer. These include the use of viral based vectors (e.g., retroviruses, adenoviruses, and adeno-associated viruses) (Drumm, M. L. et al., Rosenfeld, M. A. et al., 1992; and Muzyczka, 1992), charge associating the DNA with an asialorosomucoid/poly L-lysine complex (Wilson, J. M. et al. 1992), charge associating the DNA with cationic liposomes (Brigham, K. L. et al., 1993) and the use of cationic liposomes in association with a poly-L-lysine antibody complex (Trubetskoy, V. S. et al., 1993).

[0171] Compositions Comprising Vectors of the Invention

[0172] Although non-viral based transfection systems have not exhibited the efficiency of viral vectors, they have received significant attention, in both in vitro and in vivo research, because of their theoretical safety when compared to viral vectors. Synthetic cationic molecules, have been reported which reportedly “coat” the nucleic acid through the interaction of the cationic sites on the transfection agent and the anionic sites on the nucleic acid. The positively charged coating reportedly interacts with the negatively charged cell membrane to facilitate the passage of the nucleic acid through the cell membrane by non-specific endocytosis. (Schofield, 1995) These compounds have, however, exhibited considerable sensitivity to natural serum inhibition, which has probably limited their efficiency in vivo as gene transfection agents. (Behr 1994)

[0173] A number of attempts have been made to improve the efficiency of lipid-like cationic transfection agents, some involving the use of polycationic molecules. For example, several transfection agents have been developed that contain the polycationic compound spermine covalently attached to a lipid carrier. (Behr, 1994), discloses a lipopolyamine and shows it to be more efficient at transfecting cells than single charge molecules (albeit still less efficient than viral vectors). The agent reported by Behr was, however, toxic, and caused cell death.

[0174] A few such lipid delivery systems for transporting DNA, proteins, and other chemical materials across membrane boundaries have been synthesized by research groups and business entities. Most of the synthesis schemes are relatively complex and generate lipid based delivery systems having only limited transfection abilities. A need exists in the field of gene therapy for cationic lipid species that have a high biopolymer transport efficiency. It has been known for some time that a very limited number of certain quaternary ammonium derivatized (cationic) liposomes spontaneously associate with DNA, fuse with cell membranes, and deliver the DNA into the cytoplasm (as noted above, these species have been termed “cytofectins”). LIPOFECTIN TM. represents a first generation of cationic liposome formulation development. LIPOFECTIN TM is composed of a 1:1 formulation of the quaternary ammonium containing compound DOTMA and dioleoylphosphatidylethanolamine sonicated into small unilamellar vesicles in water. Problems associated with LIPOCFECTIN TM include non-metabolizable ether bonds, inhibition of protein kinase C activity, and direct cytotoxicity. In response to these problems, a number of other related compounds have been developed. The monoammonium compounds of the subject invention improve upon the capabilities of existing cationic liposomes and serve as a very efficient delivery system for biologically active chemicals.

[0175] Most Preferred Vectors of the Invention

[0176] Most preferred recombinant vectors according to the invention include pASΔΔ(FIG. 2), pACTIIst (FIG. 3), pT18 (FIG. 4), pUT18C (FIG. 5), pT25 (FIG. 6), pKT25(FIG. 7), pB5 (FIG. 12) and pP6 (FIG. 13) containing inserted therein a nucleic acid encoding a Selected Interacting Domain (SID®) polypeptide or a variant thereof as defined above.

[0177] The present invention is also directed to a vector usable in a two-hybrid method which consists of the vector pP6 which is shown in FIG. 13. As disclosed in example 1, the vector pP6 has been successfully used for preparing a collection of recombinant plasmids consisting of a genomic DNA library from the pathogenic strain H77 of the hepatitis C virus.

[0178] The invention also pertains to a vector usable in two-hybrid method which consists of the vector pB5. As disclosed in example 1, the vector pB5 has been successfully used in a yeast two hybrid method as a bait plasmid.

Recombinant Cell Hosts

[0179] In one embodiment, a Selected Interacting Domain (SID®) polypeptide of the invention or a variant thereof is recombinantly produced in a desired host cell which has been transfected or transformed with a nucleic acid encoding said Selected Interacting Domain (SID®) polypeptide or with a recombinant vector as defined above within which a nucleic acid encoding a Selected Interacting Domain (SID®) polypeptide of the invention is inserted.

[0180] Recombinant cell hosts are another aspect of the present invention.

[0181] Such cell hosts generally comprise at least one copy of a nucleic acid encoding a Selected Interacting Domain (SID®) polypeptide of the invention or a variant thereof

[0182] Preferred cells for expression purposes will be selected in function of the objective which is sought. For example, in the embodiment wherein the production of a Selected Interacting Domain (SID®) polypeptide according to the invention in large quantities is sought, the nature of the host cell used for its production is relatively indifferent, provided that large amounts of Selected Interacting Domain (SID®) polypeptides of the invention are produced and that optional further purification steps may be carried out easily.

[0183] However, in the embodiment wherein the Selected Interacting Domain (SID®) polypeptide is recombinantly produced within a host organism for the purpose of interfering with a specific protein-protein interaction, then the host organism is selected among the host organisms which are suspected to produce naturally said polypeptide of interest.

[0184] Consequently, mammalian and typically human cells, as well as bacterial, yeast, fungal, insect, nematode and plant cells are cell hosts encompassed by the invention and which may be transfected either by a nucleic acid or a recombinant vector as defined above.

[0185] Examples of suitable recombinant host cells include VERO cells, HELA cells (e.g. ATCC N^(o)CCL2), CHO cell-lines (e.g. ATCC N^(o)CCL61) COS cells (e.g. COS-7 cells; COS cell referred to ATCC N^(o)CRL1650), W138, BHK, HepG2, 3T3 (e.g. ATCC N^(o)CRL6361), A549, PC12, K562 cells, 293 cells, Sf9 cells (e.g. ATCC N^(o)CRL1711) and Cv1 cells (e.g. ATCC N^(o)CCL70).

[0186] Other suitable host cells are usable according to the invention include prokaryotic host cells strains of Escherichia coli (e.g. strain DH5-α), of Bacillus subtilis, of Salmonella typhimurium, or strains of genera such as Pseudomonas, Streptomyces and Staphylococcus.

[0187] Further suitable host cells usable according to the invention include yeast cells such as those of Saccharomyces, typically Saccharomyces cerevisiae.

[0188] The invention also relates to a method for producing a SID® polypeptide as defined above, wherein said method comprises the steps of:

[0189] a) cultivating a cell host which has been transformed with a SID® nucleic acid of the invention or with a vector containing a SID® nucleic acid in an appropriate culture medium;

[0190] b) recovering the SID® recombinant polypeptide from the culture supernatant or from the cell lysate.

[0191] The SID® polypeptides or variant thereof thus recombinantly obtained may be purified, for example by high performance liquid chromatography, such as reverse phase and/or cationic exchange HPLC, as described by ROUGEOT et al. (1994). The reason to prefer this kind of peptide or protein purification is the lack of by-products found in the elution samples which renders the resultant purified protein more suitable for a therapeutic use.

Two-hybrid Methods of the Invention

[0192] a) Yeast Two-hybrid Methods

[0193] The invention also pertains to a yeast two-hybrid method for selecting a recombinant cell clone containing a vector comprising a nucleic acid insert encoding a prey polypeptide which binds with a SID® polypeptide of SEQ ID N^(o)1 to 38 or a variant thereof, wherein said method comprises the steps of:

[0194] a) mating at least one first recombinant yeast cell clone of a collection of recombinant yeast cell clones transformed with a plasmid containing the prey polynucleotide to be assayed with a second aploïd recombinant Saccharomyces cerevisiae cell clone transformed with a plasmid containing a bait polynucleotide encoding a SID® polypeptide of the invention or a variant thereof;

[0195] b) cultivating diploid cells obtained in step a) on a selective medium; and

[0196] c) selecting recombinant cell clones which grow on said selective medium.

[0197] The yeast two-hybrid method above may further comprise the step of:

[0198] d) characterizing the prey polynucleotide contained in each recombinant cell clone selected in step c).

[0199] Most preferably, such a yeast two-hybrid method may be performed by the one skilled in the art as it is disclosed in example 2 hereafter.

[0200] According to the yeast two-hybrid method above, a SID® polypeptide of the invention or a variant thereof is used as a bait polypeptide.

[0201] In a preferred embodiment of the yeast two-hybrid method described above, the prey polynucleotide is a DNA fragment from the genome of a pathogenic strain of the hepatitis C virus (HCV) ranging from about 150 to about 600 nucleotides in length and which is inserted in a vector which is contained in one recombinant clone of a collection of recombinant cell clones.

[0202] b) Bacterial Two-hybrid Method

[0203] A bacterial two-hybrid method of the invention may be performed by the one skilled in the art according to the teachings of KARIMOVA et al. (1998).

[0204] The first step of selecting a collection of nucleic acids encoding polypeptides which binds specifically to the bait polypeptide may also be carried out through a bacterial two-hybrid system.

[0205] According to such bacterial two-hybrid system, bacterial cell clones, preferably Escherichia coli cells, are transformed with a plasmid containing a bait polynucleotide encoding a bait polypeptide.

[0206] Then, plasmids containing a DNA insert are provided by rescuing the plasmids obtained from the collection of yeast clones containing the genomic DNA or cDNA library which are described in the previous section entitled “Yeast two-hybrid system”. For example, the plasmid rescue may be carried out according to the following steps:

[0207] (i) extracting plasmid DNA contained in the collection of yeast clones obtained as disclosed in the previous section, by using a conventional DNA extraction buffer and a phenol: chloroform: isoamyl alcohol (25:24:1) before centrifuging;

[0208] (ii) transferring a desired volume of the supernatant obtained at the end of step (i) to a sterile Eppendorf tube and add a precipitation buffer (ethanol/NH₄Ac) before centrifuging and resuspending the pellet after washing in ethanol;

[0209] (iii) transforming Escherichia coli cells (e.g. Escherichia coli cells of strain NC 1066) which have been rendered electrocompetent with a desired volume (e.g. 1 μl) of the yeast plasmid DNA extract obtained at step (ii) by electroporation;

[0210] (iv) collecting the transformed Escherichia coli cells.

[0211] Alternatively, a collection of Escherichia coli cell clones containing a collection of HCV genomic DNA inserts may be obtained by constructing the DNA library directly in the bacterial cell, such as disclosed in Flajolet et al. (2000).

[0212] Then, the bacterial recombinant cells which have been transformed both with a plasmid containing a bait polynucleotide encoding a bait polypeptide and a plasmid containing a prey polynucleotide encoding a prey polynucleotide is cultivated on a selective medium.

[0213] Then, recombinant cell clones capable of growing on said selective medium are selected and the DNA inserts of the plasmids containing therein are sequenced.

[0214] By bacterial two-hybrid system is generally intended a method that usually makes use of at least one reporter gene, the transcription of which is activated when a prey polypeptide and a bait polypeptide produced by the recombinant cell due to the triggering of the transcription of said at least one reporter gene when both the specific domain contained in one prey polypeptide and the complementary domain contained in the bait polypeptide are binding one to the other.

[0215] The invention further pertains to a bacterial two-hybrid method for identifying a recombinant cell clone containing a prey polynucleotide encoding a prey polypeptide which binds with a SID® polypeptide of SEQ ID N^(o)1 to 38 or a variant thereof, wherein said method comprises the steps of:

[0216] a) transforming bacterial cell clones with a plasmid containing a SID® polynucleotide encoding a SID® polypeptide of the invention or a variant thereof;

[0217] b) rescuing prey plasmids containing prey polynucleotides wherein each prey polynucleotide is a DNA fragment from the genome of a desired organism and wherein each prey plasmid is contained in one recombinant yeast cell clone of a collection of recombinant yeast cell clones;

[0218] c) transforming the recombinant bacterial cell clones obtained in step a) with the plasmids rescued in step b);

[0219] d) cultivating bacterial recombinant cells obtained in step c) on a selective medium; and

[0220] e) selecting recombinant cell clones which grow on said selective medium.

[0221] The bacterial two-hybrid system described above may further comprise the step of f) characterizing the prey polynucleotide contained in each recombinant cell clone selected at step e).

[0222] In one preferred embodiment of the yeast or bacterial two-hybrid methods described above, the prey polypeptide is a human polypeptide expressed by a mammal which is infected by the Hepatitis C virus, like human and monkeys, typically chimpanzees.

[0223] Generally, the yeast two-hybrid method or the bacterial two-hybrid method as disclosed herein may be performed with prey polypeptides of any origin, either of viral, fungal, bacterial or mammal origin, i.e. either of prokaryotic or eukaryotic origin.

[0224] In a second preferred embodiment of the two-hybrid methods above, the prey polypeptide is an HCV polypeptide.

[0225] Most preferably, the prey polypeptide is encoded by a strain of the hepatitis C virus which is pathogenic for human, such as strain H77.

Sets of Nucleic Acids and Sets of Polypeptides of the Invention

[0226] In yet another aspect, the present invention relates to a set of two nucleic acids consisting of:

[0227] i) a first nucleic acid encoding a SID® polypeptide of SEQ ID N^(o)1 to 39 of the invention or a variant thereof; and

[0228] ii) a second nucleic acid encoding a prey polypeptide which binds specifically with a SID® polypeptide defined in i).

[0229] In still a further aspect, the invention is also directed to a set of two polypeptides consisting of:

[0230] i) a first polypeptide consisting of a SID® polypeptide of SEQ ID N^(o)1 to 39 of the invention or a variant thereof; and

[0231] ii) a second polypeptide which binds specifically with the first polypeptide.

[0232] The invention further relates to a complex formed between

[0233] i) a first polypeptide consisting of a SID® polypeptide of SEQ ID N^(o)1 to N^(o)38 of the invention; and

[0234] ii) a second polypeptide which binds specifically with the first polypeptide.

[0235] The invention also relates to a protein-protein interaction wherein the two interacting proteins consist of a set of two polypeptides as defined above.

[0236] In a preferred embodiment, the invention relates to the protein-protein interactions wherein the sets of two polypeptides consist of a SID®polypeptide of SEQ ID N^(o)1 to 38 and an HCV polypeptide.

[0237] When several reiterations of the two-hybrid method are performed and thus common SID® polypeptide and prey polypeptides are selected, a map of all the interactions between these polypeptides may be designed, that take into account of the known and/or suspected biological function of each of the interacting polypeptides.

[0238] Table 1 illustrates protein-protein interaction between the SID® polypeptides of SEQ ID N^(o)1 to 38 and polypeptides of SEQ ID N^(o)77 to 113 which are encoded by the genome of strain H77 of the hepatitis C virus which is pathogenic for a mammal, like human or chimpanzee.

[0239] Thus, the data presented in table 1 disclose particular sets of nucleic acids as well as particular sets of polypeptides which are encompassed by the present invention.

[0240] For example, table 1 discloses that the nucleic acid of SEQ ID N^(o)39 encodes the SID® polypeptide of SEQ ID N^(o)1 which contains exclusively (100%) an aminoacid sequence from the Core protein of HCV strain H77.

[0241] The nucleic acid of SEQ ID N^(o)39 starts at the nucleotide in position 446 and ends at the nucleotide in position 600 of the HCV genome which is described by YANAGI et al. (1997).

[0242] Table 1 also discloses that the SID® polypeptide of SEQ ID N^(o)1 is part of a set of polypeptides of the invention, wherein the second polypeptide of said set of polypeptides consists of the polypeptide of SEQ ID N^(o)77 which is encoded by the nucleic acid sequence of SEQ ID N^(o)114, which nucleic acid sequence has 87% of its sequence which is derived from the region of the H77 strain HCV DNA encoding the Core protein.

[0243] Thus , a particular set of polypeptides according to the invention consists of:

[0244] i) the polypeptide of SEQ ID N^(o)1; and

[0245] ii) the polypeptide of SEQ ID N^(o)77.

[0246] The same reasoning apply for every set of polypeptides disclosed in table 1, which are expressly part of the present invention.

[0247] Similarly, a particular set of nucleic acids according to the invention consists of.

[0248] (i) the nucleic acid of SEQ ID N^(o)39; and

[0249] (ii) the nucleic acid of SEQ ID N^(o)114.

[0250] The same reasoning apply for every set of nucleic acids disclosed in table 1, which are expressly part of the present invention.

[0251] Thus, particular sets of two polypeptides of the invention are respectively SEQ ID N^(o)77/SEQ ID N^(o)1; SEQ ID N^(o)78/SEQ ID N^(o)2; SEQ ID N^(o)78/SEQ ID N^(o)3; SEQ ID N^(o)79/SEQ ID N^(o)4; SEQ ID N^(o)80/SEQ ID N^(o)5; SEQ ID N^(o)81/SEQ ID N^(o)6; SEQ ID N^(o)82/SEQ ID N^(o)7; SEQ ID N^(o)83/SEQ ID N^(o)8; SEQ ID N^(o)84/SEQ ID N^(o)9; SEQ ID N^(o)85/SEQ ID N^(o)10; SEQ ID N^(o)86/SEQ ID N^(o)11; SEQ ID N^(o)87/SEQ ID N^(o)12; SEQ ID N^(o)88/SEQ ID N^(o)13; SEQ ID N^(o)89/SEQ ID N^(o)14; SEQ ID N^(o)90/SEQ ID N^(o)15; SEQ ID N^(o)91/SEQ ID N^(o)16; SEQ ID N^(o)92/SEQ ID N^(o)17; SEQ ID N^(o)93/SEQ ID N^(o)18; SEQ ID N^(o)94/SEQ ID N^(o)19; SEQ ID N^(o)95/SEQ ID N^(o)20; SEQ ID N^(o)96/SEQ ID N^(o)21; SEQ ID N^(o)97/SEQ ID N^(o)22; SEQ ID N^(o)98/SEQ ID N^(o)23; SEQ ID N^(o)99/SEQ ID N^(o)24; SEQ ID N^(o)100/SEQ ID N^(o)25. SEQ ID N^(o)101/SEQ ID N^(o)26. SEQ ID N^(o)102/SEQ ID N^(o)27; SEQ ID N^(o)103/SEQ ID N^(o)28. SEQ ID N^(o)104/SEQ ID N^(o)29; SEQ ID N^(o)105/SEQ ID N^(o)30; SEQ ID N^(o)106/SEQ ID N^(o)31; SEQ ID N^(o)107/SEQ ID N^(o)32; SEQ ID N^(o)108/SEQ ID N^(o)33; SEQ ID N^(o)109/SEQ ID N^(o)34; SEQ ID N^(o)110/SEQ ID N^(o)35; SEQ ID N^(o)111/SEQ ID N^(o)36; SEQ ID N^(o)112/SEQ ID N^(o)37; and SEQ ID N^(o)113/SEQ ID N^(o)38.

[0252] Similarly, particular sets of two nucleic acids according to the invention are respectively: SEQ ID N^(o)114/SEQ ID N^(o)39; SEQ ID N^(o)115/SEQ ID N^(o)40; SEQ ID N^(o)115/SEQ ID N^(o)41; SEQ ID N^(o)116/SEQ ID N^(o)42; SEQ ID N^(o)117/SEQ ID N^(o)43; SEQ ID N^(o)118/SEQ ID N^(o)44; SEQ ID N^(o)119/SEQ ID N^(o)45; SEQ ID N^(o)120/SEQ ID N^(o)46; SEQ ID N^(o)121/SEQ ID N^(o)47; SEQ ID N^(o)122/SEQ ID N^(o)48; SEQ ID N^(o)123/SEQ ID N^(o)49; SEQ ID N^(o)124/SEQ ID N^(o)50; SEQ ID N^(o)125/SEQ ID N^(o)51; SEQ ID N^(o)126/SEQ ID N^(o)52; SEQ ID N^(o)127/SEQ ID N^(o)53; SEQ ID N^(o)128/SEQ ID N^(o)54; SEQ ID N^(o)129/SEQ ID N^(o)55; SEQ ID N^(o)130/SEQ ID N^(o)56; SEQ ID N^(o)131/SEQ ID N^(o)57; SEQ ID N^(o)132/SEQ ID N^(o)58; SEQ ID N^(o)133/SEQ ID N^(o)59; SEQ ID N^(o)134/SEQ ID N^(o)60; SEQ ID N^(o)135/SEQ ID N^(o)61; SEQ ID N^(o)136/SEQ ID N^(o)62; SEQ ID N^(o)137/SEQ ID N^(o)63; SEQ ID N^(o)138/SEQ ID N^(o)64; SEQ ID N^(o)139/SEQ ID N^(o)65; SEQ ID N^(o)140/SEQ ID N^(o)66; SEQ ID N^(o)141/SEQ ID N^(o)67; SEQ ID N^(o)142/SEQ ID N^(o)68; SEQ ID N^(o)143/SEQ ID N^(o)69; SEQ ID N^(o)144/SEQ ID N^(o)70. SEQ ID N^(o)145/SEQ ID N^(o)71; SEQ ID N^(o)146/SEQ ID N^(o)72. SEQ ID N^(o)147/SEQ ID N^(o)73; SEQ ID N^(o)148/SEQ ID N^(o)74; SEQ ID N^(o)149/SEQ ID N^(o)75 and SEQ ID N^(o)150/SEQ ID N^(o)76.

[0253] The protein-protein interactions disclosed in table 1 allows the design of a map of interactions between various polypeptides encoded by the genome of the H77 strain of HCV.

[0254] In such a Protein Interaction Map (PIM®) wherein each SID® polypeptide is linked to the bait polypeptide onto which it specifically binds, for example by an arrow.

[0255] Such a Protein Interaction Map (PIM®) may help the one skilled in the art to decipher a whole metabolical and/or physiological pathway that is functionally active within a pathogenic strain of HCV. Protein Interaction Map and computable version of PIM® are part of the present invention.

[0256] Therefore, in still another aspect, the present invention is directed to a computable readable medium (such as floppy disk, CD-ROM and all electronic or magnetic format which can be read by a computer) having stored thereon protein-protein interactions according to the invention, preferably stored in a form of a Protein Interaction MAP, as shown, for example, in FROMONT-RACINE et al. (1997).

[0257] In a preferred embodiment, the invention comprises a computable readable medium as defined above, wherein the protein-protein interactions stored thereon are linked to annotated data base, for example through Internet.

[0258] In another preferred embodiment, the invention comprises a data bank containing the protein-protein interactions stored thereon, said data bank being available on a world-wide web site.

METHODS FOR SELECTING INHIBITORS OF PROTEIN-PROTEIN INTERACTIONS OF THE INVENTION

[0259] The transformed host cells as described above can also be used as models so as to study the interactions between a SID® polypeptide of the invention and its binding partner polypeptide, or between a SID® polypeptide of the invention and chemical or protein compounds which inhibit the binding between said SID® polypeptide and its binding partner polypeptide.

[0260] Example of a SID® polypeptide and its binding partner polypeptides are typically the sets of polypeptides of the invention which are described above.

[0261] In particular, the transformed host cells of the invention may be used for the selection of molecules which interact with a SID® polypeptide as described herein, as cofactor or as inhibitor, in particular a competitive inhibitor, or alternatively having an agonist or antagonist activity on the protein-protein interaction wherein said SID® polypeptide is involved. Preferably, the said transformed host cells will be used as a model allowing, in particular, the selection of products which make it possible to prevent and/or to treat pathologies induced by the hepatitis C virus.

[0262] Consequently, the invention also consists of a method for selecting a molecule which inhibits the protein-protein interaction of a set of two polypeptides as defined above, wherein said method comprises the steps of:

[0263] a) cultivating a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two vectors wherein:

[0264] i) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said two-polypeptides and a DNA binding domain;

[0265] ii) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides and an activating domain capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting;

[0266] on a selective medium containing the molecule to be ested and allowing the growth of said recombinant host cell when the toxic reporter gene is not activated; and

[0267] b) selecting the molecule which inhibits the growth of the recombinant host cell defined in step a).

[0268] The invention is also directed to a method for selecting a molecule which inhibits the protein-protein interaction of a set of two polypeptides as defined above, wherein said method comprises the steps of:

[0269] a) cultivating a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two vectors wherein:

[0270] i) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said two polypeptides and the first domain of an enzyme;

[0271] ii) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides and the second part of said enzyme capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting, said interaction recovering the catalytic activity of the enzyme;

[0272] on a selective medium containing the molecule to be tested and allowing the growth of said recombinant host cell when the toxic gene is not activated; and

[0273] b) selecting the molecule which inhibits the growth of the recombinant host cell defined in step a).

[0274] In a preferred embodiment, said toxic reporter gene that can be used for negative selection is URA3, CYH1 or CYH2 gene.

[0275] For example, a method for the screening of a molecule which inhibits the interaction between a SID® polypeptide of the invention with its binding protein counterpart may comprise the following steps:

[0276] transform a permeabilized yeast cell with two vectors, respectively a first vector containing a SID® nucleic acid of the invention and a second vector containing a prey nucleic acid as defined in the present specification;

[0277] plate on top agar the transformed permeabilized yeast cells above on square boxes;

[0278] apply by spotting the candidate inhibitor molecules to test on top agar as soon as it is solidified;

[0279] incubates, for example, overnight at 30° C., and

[0280] select the inhibitor compounds that allow the growth of the transformed yeast cells.

[0281] The invention also provides for a kit for the screening of a molecule which inhibits the protein-protein interaction of a set of two polypeptides as defined above, wherein said kit comprises a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two vectors wherein:

[0282] i) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said two polypeptides and a DNA binding domain;

[0283] ii) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides and an activating domain capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting.

[0284] Another object of the invention consists of a kit for the screening of a molecule which inhibits the protein-protein interaction of a set of two polypeptides as defined above, wherein said kit comprises a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two plasmids wherein:

[0285] i) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said two polypeptides and the first domain of a protein;

[0286] ii) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides and the second part of said protein capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting, said interaction recovering the activity of the protein. In the selection methods above, the transcription or activating domain and the DNA-binding domain may be derived from Gal4 and LexA respectively.

[0287] In the embodiment wherein the first domain is a first part of an enzyme and a complementary domain is a second part of the same enzyme, and wherein the proximity of the two parts of the enzyme restores the enzyme activity and activates a reporter gene, the two parts of the enzymes are most preferably the T25 and T18 polypeptides that form the catalytic domain of the Bordetella pertussis adenylate cyclase.

[0288] As an illustrative embodiment, the reporter gene is chosen among the group consisting of a nutritional gene or also a gene the expression of which is visualised by colorimetry such as His3, LacZ or both LacZ and His3.

Market Compounds of the Invention

[0289] The Selected Interacting Domain (SID®) polypeptides of SEQ ID N^(o)1 to 38 of the invention and variants thereof defined in the present specification, and which bind specifically to a polypeptide of interest (e.g. a bait polypeptide), are useful as reagents for detecting, labelling, targeting or purifying specifically a polypeptide of interest, typically a polypeptide encoded by HCV, within a sample, since the SID® polypeptides possess properties that have never been reached using conventional detection compounds, such as those of an antibody or an antibody fragment.

[0290] Firstly, the SID® polypeptides of the invention possess a high specificity of binding to the polypeptide of interest, since a SID® polypeptide consists of a portion of a larger polypeptide which binds in a highly specific manner to the polypeptide of interest in the natural environment within the eukaryotic cell infected by the Hepatitis C virus.

[0291] Secondly, the SID® polypeptide generally has a low molecular weight, generally from 3 kDa, and are thus easy to produce, on the one hand, and, on the other hand, can be easily introduced within a cell when the detection of the localisation or of the expression of the polypeptide of interest is sought. Moreover, the small size of a SID® polypeptide allows its passage through inner cell barriers such as the nucleus membrane, or the membranes surrounding the different cell organites.

[0292] Thus, a first object of the invention consists of a marker compound wherein said compound comprises:

[0293] a) a Selected Interacting Domain (SID®) polypeptide of the invention or a variant thereof that binds specifically to the polypeptide of interest; and

[0294] b) a detectable molecule bound thereto.

[0295] Such a marker compound is primarily useful for detecting, labelling or targeting a polypeptide of interest, for example a polypeptide of interest contained in a sample.

[0296] A detectable molecule according to the invention comprises, or alternatively consists of, any molecule which produces or can be induced to produce a signal. The detectable molecule can be a member of the signal producing system that includes the signal producing means .

[0297] The detectable molecule may be isotopic or non-isotopic. By way of example and not limitation, the detectable molecule can be part of a catalytic reaction system such as enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, co-enzymes, or catalysts. Part of a chromogen system such as fluorophores, dyes, chemiluminescers, luminescers, or sensitizers. A dispersible particle that can be non-magnetic or magnetic, a solid support, a liposome, a ligand, a receptor, a hapten radioactive isotope, and soforth.

[0298] It must be generally understood that the whole embodiments disclosed in the present specification involving a Selected Interacting Domain (SID®) polypeptide is straightfully applied also to any variant thereof.

[0299] Fluorescent Detectable Molecules

[0300] In one aspect of the marker compound according to the invention, the detectable molecule consists of a fluorescent molecule. Fluorescent moieties which are frequently used as labels are for example those described by Ichinose et al. (1991). Other fluorescent detectable molecules are fluorescing isothiocyanate (FITC) such as described by Shattil et al. (1987) or by Goding et al. (1986). The fluorescent detectable molecule may also comprise a phycoerythrin as taught by Goding et al. (1986), and Shattil et al. (1985). Other examples of fluorescent detectable molecules suitable for use as labels of a marker compound according to the invention are rhodamine isothiocyanate, dansyl chloride and XRITC.

[0301] Another fluorescent detectable molecule consists of the green fluorescent protein (GFP) of the jelly fish Aequorea victoria, and their numerous fluorescent protein derivatives.

[0302] The one skilled in the art may advantageously refer to the articles of CHALFIE et al. (1994) and of HEIM et al. (1994) which discloses the uses of GFP for the study of gene expression and protein localisation. The one skilled in the art may also refer to the article of Rizzuto et al. (1995), which discusses the use of wild-type GFP as a tool for visualising subcellular organelles in cells, to the article of KAETHER and GERDES (1995), which reports the visualisation of protein transport along the secretary passway using wild-type GFP, the article of HU and CHENG (1995), which relates to the expression of GFP in plant cells and also to the article of Davis et al. (1995) which discloses the GFP expression in drosophilia embryos. For the use of several fluorescent variants of GFP, the one skilled in the art may refer to the article of Delagrave et al. (1995), as well as to the article of Heim et al. (1995). DNA encoding GFP is available commercially, for example from Clontech in Palo Alto, Calif., USA. The one skilled in the art may use also humanized GFP genes such as those described in the U.S. Pat. No. 6,020,192 and also the GFP protein disclosed in the U.S. Pat. No. 5,941,084.

[0303] Another fluorescent protein that may be used in a marker compound according to the invention consists of the yellow fluorescent protein (YFP).

[0304] A further suitable luminescent protein consists of the luciferase protein.

[0305] Detectable Molecules Exhibiting a Catalytic Activity

[0306] In another embodiment of a detectable molecule included in a marker compound according to the invention, said detectable molecule is endowed with a catalytic activity and may thus consists of enzymes and catalytically active enzyme fragments. Some enzymatic labels are described in U.S. Pat. No. 3,654,090. Such enzymes may be for example horse radish peroxydase (HRP), alkaline phosphatase or glutathione peroxydase which are well known from the one skilled in the art.

[0307] Enzymes, enzyme fragments, enzyme inhibitors, enzyme substrates, and other components of enzyme reaction systems can be used as detectable molecules. Where any of these components is used as a detectable molecule, a chemical reaction involving one of the components is part of the signal producing system.

[0308] Coupled catalysts can also involve an enzyme with a non-enzymatic catalyst. The enzyme can produce a reactant, which undergoes a reaction catalysed by the non-enzymatic catalyst or the non-enzymatic catalyst may produce a substrate (including co-enzymes) for the enzyme. The one skilled in the art may advantageously refer to the U.S. Pat. No. 4,160,645 which disclose a white variety of non enzymatic catalysts, which may be employed, the appropriate portions of which are incorporated therein by reference.

[0309] The enzyme or co-enzyme employed provides the desired amplification by producing a product, which absorbs light, e.g., a tye, or emits lights upon irradiation, e.g., a fluorescer. Alternatively, the catalytic reaction can lead to direct light emission, e.g., chemiluminescence. A large number of enzymes and co-enzymes for providing such products are described in the U.S. Pat. No. 4,275,149, columns 19 to 23 and U.S. Pat. No. 4,318,980, columns 10 to 14 which disclosures are incorporated herein by reference.

[0310] A number of enzyme combinations are set forth in U.S. Pat. No. 4,275,149, columns 23 to 28 which disclosures are incorporated herein by reference.

[0311] When a single enzyme is used as the detectable molecule, or alternatively as comprised in the detectable molecule, such enzymes may find use are hydrolases, transferases, lyases, isomerases, ligases or synthetases and oxydoreductases.

[0312] Alternatively, luciferases may be used such as firefly luciferase and bacterial luciferase.

[0313] Primarily, the enzymes of choice, based on the I.U.B. classification are: (i) class 1. Oxydoreductases and (ii) class 3. Hydrolases. Most preferred oxydoreductases are (i) dehydrogenases of class 1.1, more particularly 1.1.1, 1.1.3. and 1.1.99 and (ii) peroxydases in class 1.11. of the hydrolases, particularly class 3.1., more particularly 3.1.3 and class 3.2, more particularly 3.2.1. are preferred.

[0314] Illustrative dehydrogenases include malate dehydrogenase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase. Of the oxydases, glucose oxydases is exemplary. Of the peroxydases, horse radish peroxydase is illustrative. Of the hydrolases, alkaline phosphatases, β-glucosydase and lysozyme are illustrative.

[0315] Chemiluminescent Detectable Molecules

[0316] The detectable molecule comprised within the marker compound according to the invention may also consist in a chemiluminescent moiety. The chemiluminescent source involves a compound, which becomes electronically excited by a chemical reaction and may emit light which serves at as the detectable signal or donates energy to a fluorescent acceptor.

[0317] A diverse number of families of compounds have been found to provide chemiluminescent under a variety of conditions. When family of compounds is 2,3-dihydro-1,4-phtalazinedinone. The most utilised compound is luminol, which is the 5-amino analogue of the compound above. Other members of the family include the 5-amino-6,7,8-trimethoxy-and the dimethylamine-[ca]benzo analogue. These compounds can be made to luminance with alkaline hydrogen peroxyde or calcium hypochlorite and base.

[0318] Another family of compounds is the 2,4,5-triphenylimidazoles, with lophine as the common name for the parent product. Chemiluminescent analogues include para-dimethylamino- and para-methoxy-substituents. Chemiluminescents may also be obtained with geridinium esters, dioxetanes and oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins.

[0319] Radioactive Detectable Molecules

[0320] In a further embodiment of a detectable molecule comprised in a marker compound according to the invention, said detectable molecule is radio-actively labelled such as with [³H], [³²P], [and [¹²⁵I].

[0321] Colloïdal Metal Detectable Molecules

[0322] In still a further embodiment, the detectable molecule comprised in a marker compound according to the invention may include a colloïdal metal particle. Colloïdal metals have been employed in immuno assays previously. Mostly, they consisted of either colloïdal iron or gold. The one skilled in the art may advantageously refer to the articles of Horisberger (1981) and Martin et al. (1990). In other case, the metals are chosen for their colour, i.e., their presence is determined by their colour or electron density under an electron microscope. Both the colour and electron density are directly proportional to the mass of the metal colloïd.

Structure of the Marker Compounds of the Invention

[0323] In a first preferred embodiment of a marker compound of the invention, the detectable molecule is covalently bound to the Selected Interacting Domain (SID®) polypeptide of SEQ ID N^(o)1 to SEQ ID N^(o)38 or a variant thereof.

[0324] According to this specific embodiment, detectable molecules comprising fluorescent proteins such as GFP and YFP, enzymes or enzyme fragments such as alkaline phosphatase, glutathione peroxydase and horse radish peroxydase, chemiluminescent molecules, radioactive labels or colloidal metal particles will be preferred.

[0325] General methods that may be used by the one skilled in the art for covalently binding the detectable molecules to the Selected Interacting Domain (SID®) polypeptide are described in the numerous bibliographic references related to the preparation of the antibody conjugates used for carrying out immunoassays.

[0326] In a second preferred embodiment of a marker compound according to the invention, the detectable molecule is non-covalently bound to the Selected Interacting Domain (SID®) polypeptide or a variant thereof.

[0327] In a first preferred aspect of this second preferred embodiment, the detectable molecule consists of an antibody directed specifically against the Selected Interacting Domain (SID®) polypeptide or a variant thereof.

[0328] The antibodies directed specifically against the Selected Interacting Domain (SID®) polypeptide or a variant thereof may be indifferently radioactivity or non radioactivity labelled.

Nucleic Acids Encoding a Marker Compounds of the Invention

[0329] The present invention also relates to a nucleic acid encoding a marker compound as defined above.

[0330] Most preferred nucleic acids encompassed by the invention include polynucleotides that encode a marker compound wherein the Selected Interacting Domain (SID®) polypeptide of SEQ ID N^(o)1 to 38 or a variant thereof is covalently bound to the detectable molecule and wherein the detectable molecule consists itself of a polypeptide.

[0331] Most preferred nucleic acids are those of SEQ ID N^(o)39 to 76.

[0332] In a first preferred embodiment of a nucleic acid according to the invention, said nucleic acid encodes for a Selected Interacting Domain (SID®) polypeptide which is fused to a fluorescent protein, such as GFP and YFP.

[0333] In a second preferred embodiment of a nucleic acid according to the invention, said nucleic acid encodes for a Selected Interacting Domain (SID®) polypeptide which is fused to a polypeptide endowed with a catalytic activity, such as an enzyme or an enzymatically active enzyme fragment, like alkaline phosphatase, glutathione peroxydase and horse radish peroxydase.

[0334] In a preferred embodiment, a nucleic acid encoding a marker compound of the invention comprises a DNA coding sequence which is transcribed and translated into said marker compound in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon and a translation stop codon. A coding sequence can include, but is not limited to:

[0335] prokaryotic sequences, for example when the Selected Interacting Domain (SID®) nucleic acid and the nucleic acid fused thereto which encodes the detectable molecule are of prokaryotic origin;

[0336] prokaryotic and eukaryotic sequences, for example the nucleic acid encoding the detectable molecule originates from an eukaryotic host organism.

[0337] If the coding sequence is intended for expression in an eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

[0338] In a most preferred embodiment of a nucleic acid sequence according to the invention, said nucleic acid sequence include a regulatory region which is functional in the host organism within which the expression of said nucleic acid sequence is sought, wherein said regulatory region comprises a promoter sequence.

[0339] “Regulatory region” means a nucleic acid sequence which regulates the expression of a nucleic acid. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region), or may include sequences of a different origin (responsible for expressing different proteins or even synthetic proteins). In particular, the sequences can be sequences of eukaryotic or viral genes or derived sequences which stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, enhancers, transcriptional termination sequences, signal sequences which direct the polypeptide into the secretary pathways of the target cell, and promoters.

[0340] A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0341] A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.

[0342] Most Preferred Vectors for the Expression of a Marker Compound of the Invention

[0343] Most preferred recombinant vectors for expressing a marker compound of the invention include PASΔΔ (FIG. 2), pACTIIst (FIG. 3), pT18 (FIG. 4), pUT18C (FIG. 5), pT25 (FIG. 6), pKT25 (FIG. 7), pB5 (FIG. 12) and pP6 (FIG. 13) containing inserted therein a nucleic acid encoding a Selected Interacting Domain (SID®) polypeptide as defined above or a variant thereof.

[0344] The invention also pertains to recombinant host cells transformed with a vector expressing a marker compound as defined above, more particularly a vector comprising inserted therein a nucleic acid encoding said marker compound, which is operably linked to suitable regulation signals which are functional in the host cell wherein its expression is sought.

[0345] Preferred cells for expression purposes will be selected in function of the objective which is sought. For example, in the embodiment wherein the production of a marker compound according to the invention in large quantities is sought, the nature of the cell host used for its production is relatively indifferent, provided that large amounts of Selected Interacting Domain (SID®) polypeptides or marker compounds of the invention are produced and that optional further purification steps may be carried out easily.

[0346] However, in the embodiment wherein the marker compound is recombinantly produced within a host organism for the purpose of qualitative or quantitative analysis of the polypeptide of interest onto which said marker compound specifically binds, then the host organism is selected among the host organisms which are suspected to produce naturally said polypeptide of interest.

[0347] Consequently, mammalian and human cells, as well as bacterial, yeast, fungal, insect, nematode and plant cells are cell host encompassed by the invention and which may be transfected either by a nucleic acid or a recombinant vector as defined above.

Detection Methods of the Invention

[0348] The present invention further relates to the use of a Selected Interacting (SID®) polypeptide of SEQ ID N^(o)1 to 38 or a variant thereof as well as a nucleic acid encoding it for detection purposes such as nucleic acids of SEQ ID N^(o)39 to 76. It is herein reminded that a Selected Interacting Domain (SID®) polypeptide is determined according to the ability of such a (SID®) polypeptide to bind in a highly specific manner to a given (e.g. bait) polypeptide of interest, since the aminoacid sequence of a SID® polypeptide is encoded by a nucleic acid, the nucleotide sequence of which consists of the polynucleotide sequence which is common to a collection of nucleic acid sequences encoding prey polypeptides that have been selected for their specific binding properties to a (bait) polypeptide of interest, such as explained above in the section entitled “SELECTED INTERACTING DOMAIN (SID®) POLYPEPTIDES”.

[0349] The specific properties of a Selected Interacting Domain (SID®) polypeptide for binding to a given polypeptide of interest, either a viral, yeast, fungal, bacterial, insect, plant or mammal polypeptide, including a polypeptide of human origin, allow its use as a specific ligand for said polypeptide of interest of which the detection is sought.

[0350] Therefore, the use of a Selected Interacting Domain (SID®) in any detection method known in the art and which makes use of the ability of a detection ligand to bind specifically to a molecule of interest, most preferably a polypeptide of interest, fall under the scope of the present invention.

[0351] Detection methods that make use of the recognition of a molecule of interest, most preferably a polypeptide of interest, by a detection ligand are well known in the art and are primarily illustrated by the abundant literature that relate to immunoassays, which is incorporated herein by reference in its entirety.

[0352] The one skilled in the art may particularly refer to the book of Maggio (1980) (Heterogeneous assays), the U.S. Pat. No. 3,817,837 (homogeneous Immunoassays), U.S. Pat. No. 3,993,345 (Immunofluorescense methods), U.S. Pat. No. 4,233,402 (enzyme channelling techniques), U.S. Pat. No. 3,817,837 (Enzyme multiplied immunoassay technique), U.S. Pat. No. 4,366,241 and European Patent Application N^(o)EP-A 0 143 574 (Migration type assays), U.S. Pat. No. 5,202,006, U.S. Pat. No. 5,120,413 and U.S. Pat. No. 5,145,567 (Immunofixation electrophoresis, mmunoelectrophoresis), the article of Aguzzi et al. (1977), the article of White et al. (1986), the article of Merlini et al. (1983), the U.S. Pat. No. 5,228,960 (Immunosubstraction electrophoresis), the articles of Chen et al. (1991), Nielsen et al. (1991) and the U.S. Pat. No. 5,120,413 (Capillary electrophoresis).

[0353] Acellular Detection Method of the Invention

[0354] A first detection method of the invention consists of a method for detecting a polypeptide of interest within a sample, wherein said method comprises the steps of:

[0355] a) contacting a marker compound or a plurality of marker compounds according to the invention with the sample which is suspected to contain the polypeptide of interest the detection of which is sought;

[0356] b) detecting the complexes formed between said marker compound or said plurality of marker compounds and said polypeptide of interest.

[0357] The sample which is assayed for the presence of the polypeptide of interest the detection of which is sought may be of any nature , including every sample that may be used for carrying out an immunoassay.

[0358] In a first aspect, the sample may be any biological fluid, such as blood or blood separation products (e.g. serum, plasma, buffy coat), urine, saliva, tears.

[0359] In a second aspect, the sample may be any isolated biological tissue sample, including tissue sections previously fixed for purposes of histological studies.

[0360] In a third aspect, the sample may be a culture supernatant of a cell culture and a cell lysate of cultured cells.

[0361] In a first preferred embodiment of the first detection method of the invention described above, the detection step b) consists of the measure of the fluorescence signal intrinsically emitted by the detectable molecule. It may for example be taken the advantage of SID® polypeptides or variants thereof having in their aminoacid sequence one or several tryptophan aminoacid residues.

[0362] In a second preferred embodiment of the first detection method of the invention detailed above, the detection step b) consists of submitting the detectable molecule to a source of energy at the excitation wavelength of said detectable molecule, and measuring the light emitted at the emission wavelength of said detectable molecule.

[0363] An illustrative example of this second embodiment above is when the marker compound used consists of a Selected Interacting Domain (SID®) which is bound to a fluorescent molecule, such as the fluorescent proteins GFP or YFP.

[0364] For example, in the embodiment wherein the detectable molecule of the marker compound of the invention which is used according to the first detection method above comprises, or alternatively consists of, a GFP protein, the detection step c) includes illuminating the sample tested at an emission wavelength substantially equal to 490 nm, and measuring the light emitted by the marker compound which is bound to the polypeptide of interest within the sample at an emission wavelength substantially equal to 510 nm.

[0365] Preferably, the marker compounds which are not bound to the polypeptide of interest the detection of which is sought within the sample are removed before carrying out the detection step.

[0366] In a third preferred embodiment, the detection step c) of the first detection method of the invention consists of measuring the catalytic activity of the detectable molecule. In this specific embodiment, the marker compound used in the detection method comprises a detectable molecule which comprises, or alternatively which consists of, an enzyme or a catalytically active enzyme fragment, such as already detailed in the section entitled “Marker compounds of the invention”.

[0367] In a fourth preferred embodiment, the detection step b) consists of measuring the radioactivity emitted by the detectable molecule.

[0368] The present invention further relates to a kit for detecting a polypeptide of interest within a sample, wherein said kit comprises a marker compound according to the invention.

[0369] Optionally, said detection kit further comprises the reagents necessary for carrying out the detection step b), such as a suitable substrate for the particular enzyme or a catalytically active enzyme fragment used, as well as suitable buffer solutions, which may be identical to those conventionally used for performing immunoassays.

[0370] Cellular Detection Assay Using a Recombinantly Produced Marker Compound of the Invention

[0371] As already described above, any marker compound according to the invention may be produced according to genetic engineering techniques. Particularly, nucleic acid encoding a particular marker compound which binds specifically to a polypeptide of interest the detection of which is sought may be inserted in a vector, wherein said vector may be used to transfect or transform a host organism, either a prokaryotic or an eukaryotic cell host such as defined above.

[0372] In this specific embodiment, the production of a recombinant marker compound of the invention is allowed within such a transfected or transformed host cell. Once the host cell of interest is transfected or transformed with such a recombinant vector and once the recombinant marker compound is produced within the cell host of interest, then the Selected Interacting Domain (SID®) polypeptide portion of said marker compound will be able to bind specifically to its specific target polypeptide within the cell host. In this situation, the recombinantly produced marker compound of the invention will predominantly be localised at cell sites wherein the targeted polypeptide of interest is present.

[0373] This is the purpose of the second detection method of the invention which is detailed below.

[0374] A further object of the invention consists of a method for detecting a polypeptide of interest within a prokaryotic or an eukaryotic cell host, wherein said method comprises the steps of:

[0375] a) providing a cell host to be assayed;

[0376] b) transfecting said cell host with a nucleic acid encoding a marker compound of the invention, or with a recombinant vector encoding a marker compound of the invention;

[0377] c) detecting the complexes formed between the marker compound expressed by the transfected cell host and the polypeptide of interest.

[0378] Because the Selected Interacting Domain (SID®) polypeptide which is part of a marker compound of the invention specifically binds to a polypeptide which is suspected to be naturally produced by the targeted cell host, the second detection method of the invention defined above allows a qualitative as well as a quantitative detection of this targeted polypeptide which is suspected to be naturally produced by the transfected target cell host under assay.

[0379] For example, in the embodiment within which the procedure for selecting the Selected Interacting Domain (SID®) polypeptide which is part of a marker compound of the invention includes a first step wherein a collection of clones containing nucleic acid inserts derived from a H77 strain HCV genomic DNA library is prepared, the transfection of a mammalian cell, preferably a human cell, with a vector encoding such a marker compound of the invention will allow to detect the expression of a human polypeptide naturally expressed within said mammalian host cell and which naturally interacts with the HCV viral protein from which is derived the Selected Interacting Domain (SID®) polypeptide.

[0380] The second detection method of the invention defined above firstly allows the qualitative detection of the targeted polypeptide of interest which binds specifically with the recombinantly produced marker compound of the invention, and thus permits to know in which environmental conditions or at which differentiation stage the targeted polypeptide of interest is naturally produced within the cell host transfected with a vector expressing a marker compound of the invention.

[0381] Secondly, this second detection method of the invention allows the localisation of the targeted polypeptide of interest within the interior of the cell, including localisation in the plasma membrane, cytosol, nucleus and any organelle such as ribosomes, Golgi apparatus, lysosomes, phagosomes, endoplasmic reticulum and chloroplasts.

[0382] The localisation of a targeted polypeptide of interest which is expressed within the cell host under assay according to the second detection method of the invention may be carried out by any means well known in the art, including using a confocal microscope.

[0383] Thirdly, the second detection method of the invention allows also a quantitative analysis of the expression of the targeted polypeptide of interest within the cell host under assay, since the level of the detection signal produced by the detectable molecule which is part of the marker compound will be proportional to the number of complexes formed between the cell host under assay between the targeted polypeptide of interest and the recombinantly produced marker compound of the invention.

[0384] Essentially, the one skilled in the art may refer to the section entitled “Acellular detection method of the invention” above to find the teachings necessary for performing the detection step c) of the second detection method described herein.

[0385] In a first embodiment of said second detection method of the invention, the detection step c) consists of the measure of the fluorescence signal intrinsically emitted by the detectable molecule comprised in the recombinantly expressed marker compound of the invention.

[0386] In a second preferred embodiment of the second detection method above, the detection step c) consists of submitting the detectable molecule to a source of energy at the excitation wavelength of said detectable molecule and measuring the light emitted at the emission wavelength of said detectable molecule.

[0387] In still a further embodiment of the second detection method of the invention, the detection step c) consists of measuring the catalytic activity of the detectable molecule.

[0388] In another embodiment, the detection step c) consists of measuring the radioactivity emitted by the detectable molecule.

[0389] In yet a further embodiment of the second detection method of the invention, the detection step c) allows the location of the complexes formed between the recombinantly produced marker compound and the targeted polypeptide of interest within the transfected cell host.

[0390] A further object of the invention consists of a kit for detecting a polypeptide of interest within a prokaryotic or an eukaryotic cell host, wherein said kit comprises a nucleic acid encoding a marker compound as defined herein, or a recombinant vector containing inserted therein a nucleic acid encoding a marker compound of the invention.

[0391] Optionally, the detection kit above may further comprise the reagents necessary to carry out the detection step c).

[0392] Cellular Detection Method of the Invention Using a Marker Compound which is Introduced Within a Cell Host

[0393] There is a third detection method according to the invention wherein the marker compound comprising a Selected Interacting Domain (SID®) polypeptide OF SEQ ID N^(o)1 to 38 or a variant thereof is previously produced by any means and subsequently introduced into a target cell host for the purpose of detecting a targeted polypeptide of interest which binds specifically with said Selected Interacting Domain (SID(D) polypeptide.

[0394] Thus, the invention further relates to a method for detecting a polypeptide of interest within a prokaryotic or an eukaryotic cell host, wherein said method comprises the step of:

[0395] a) providing a cell host to be assayed;

[0396] b) introducing a marker compound as defined herein within said cell host; and

[0397] c) detecting the complexes formed between the marker compound and the polypeptide of interest within the cell host.

[0398] Taking into account the low molecular weight of the Selected Interacting Domain (SID®) polypeptide selected from SEQ ID N^(o)1 to 38 which is part of a marker compound of the invention, when compared with conventional specific detection molecules such as antibodies or antibody fragments, it results that the introduction of a marker compound of the invention into the interior of a target cell host will be much more easier to perform, as compared with the introduction within a cell host of a conventional marker like a labelled antibody or a labelled antibody fragment.

[0399] According to the third detection method of the invention defined above, step b) of introducing the marker compound within the target cell host may be performed by any technique well known in the art, including electroporation, and the use of molecules that will facilitate the passage of the marker compound of the invention through the cell membranes, and typically the plasma membrane.

[0400] Such molecules that facilitate the passage of a marker compound of the invention through cell membranes include, but are not limited to, penetratin, like penetratin 1.RTM (Encor, Gaithersburg, Md.), Antenna Pediae protein, cationic lipids and cationic polyacrylates.

[0401] Permeation enhancers which may be employed include bile salts such as sodium glycocholate and other molecules such as β-cyclodextrin. Bile salts are known to increase the absorption of macromolecules across membranes (Pontiroli et al., 1987).

[0402] As already detailed for the second detection method of the invention described in the previous section, the third detection method of the invention allows also the localisation of the targeted polypeptide of interest which is expressed by the cell host under assay, as well as the qualitative and quantitative analysis of the expression of said target polypeptide of interest.

[0403] The detection step c) according to the third detection method of the invention described above may be carried out in the same way than the detection step c) of anyone of the first detection method and the second detection method detailed in the previous sections herein.

[0404] In a first embodiment of the third detection method above, the detection step c) consists of the measure of the fluorescence signal intrinsically emitted by the detectable molecule.

[0405] In a second embodiment, the detection step c) consists of submitting the detectable molecule to a source of energy at the excitation wavelength of said detectable molecule and measuring the light emitted at the emission wavelength of said detectable molecule.

[0406] In a third embodiment, the detection step c) consists of measuring the catalytic activity of the detectable molecule.

[0407] In a fourth embodiment, the detection step c) consists of measuring the radioactivity emitted by the detectable molecule.

[0408] In a fifth embodiment of the third detection method of the invention, the detection step c) allows the location of the complexes formed between the marker compound and the polypeptide of interest within the target cell host under assay.

[0409] A further object of the invention consists of a kit for detecting a polypeptide of interest within a prokaryotic or an eukaryotic cell host, wherein said kit comprises a marker compound as defined herein.

[0410] The detection kit above may further comprise the reagents necessary to carry out the detection step c).

[0411] The detection kit above may also further comprise the reagents necessary to facilitate the introduction of the marker compound within the target cell host under assay.

Solid Phase Detection Method Using a Selected Interacting Domain (SID®) Polypeptide

[0412] In a further aspect of the invention, the use of a Selected Interacting Domain (SID®) polypeptide of SEQ ID N^(o)1 to 38or a variant thereof for detection purpose include a step wherein said Selected Interacting Domain (SID®) polypeptide is immobilised on a suitable substrate before bringing a sample to be assayed in contact with the substrate onto which said Selected Interacting Domain (SID®) polypeptide has been previously immobilised.

[0413] A subsequent step will consist in detecting the complexes formed between the Selected Interacting Domain (SID®) polypeptide immobilised on the substrate and the targeted polypeptide of interest the presence of which is suspected in the sample assayed.

[0414] Thus, the invention also pertains to a fourth detection method which consists of a method for detecting a polypeptide or a plurality of polypeptides of interest within a sample, wherein said method comprises the steps of:

[0415] a) providing a substrate onto which a Selected Interacting Domain (SID®) polypeptide or a plurality of Selected Interacting Domain (SID®D) polypeptides is (are) immobilised;

[0416] b) bringing into contact the substrate defined in a) with the sample to be assayed;

[0417] c) detecting the complexes formed between the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides and the target polypeptide or the plurality of target polypeptides contained in the sample.

[0418] Substrates, supports or surfaces for immobilising protein molecules are well known in the art, and a lot of them have been described for performing solid phase immunoassays.

[0419] Preferably, a plurality of Selected Interacting Domain (SID®) polypeptides of different aminoacid sequences choosen among the sequences SEQ ID N^(o)1 to 38 are immobilised on the substrate used according to the fourth detection method of the invention.

[0420] For example, a complete collection of Selected Interacting Domain (SID®) polypeptides which have been determined according to the methods described in the section entitled “Selected Interacting Domain (SID®) polypeptides” above, using nucleic acids derived from the H77 strain HCV genomic DNA as starting material, may be used for being immobilised on a suitable substrate.

[0421] According to this embodiment, the collection of Selected Interacting Domain (SID®) polypeptides of SEQ ID N^(o)1 to 38 are immobilised on the substrate in another manner, thus forming an ordered area of SID® polypeptides immobilised at known locations of the surface of said substrate.

[0422] The substrate, support or surface may be a porous or a non-porous water insoluble material. The support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulphate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers; synthetic or modified naturally occurring polymers, such as nitro-cellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide , cross-linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephtalate), nylon, poly(vinyl butyrate), said materials being used by themselves or in conjunction with other materials; glass available as Bioglass, ceramic metals and the like.

[0423] An ordered area onto which a plurality of Selected Interacting Domain (SID®) polypeptides are immobilised may be manufactured according to the techniques disclosed in the U.S. Pat. No. 5,143,854 or the PCT Application No. WO 92/10092, incorporated herein by reference for all purposes. The combination of photolithographic and fabrication techniques may, for example, enable each Selected Interacting Domain (SID®) polypeptide to occupy a very small area (“site”) on the support. In some embodiments, the site may be as small as few microns or even a single Selected Interacting Domain (SID®) polypeptide.

[0424] In a first embodiment of the fourth detection method detailed above, the plurality of Selected Interacting Domain (SID®) polypeptides are immobilized on the substrate in an order manner.

[0425] In a second embodiment of Selected interacting Domain (SID®), the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides are covalently bound to the substrate.

[0426] In a third embodiment of said method, the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides are non-covalently bound to the substrate. According to this specific embodiment, the Selected Interacting Domain (SID(®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides are covalently bound to a first ligand molecule and the substrate is coated with a second ligand molecule, wherein said second ligand molecule specifically binds to the first ligand molecule. According to such a specific embodiment, the first ligand may be biotin in which case the second ligand is most preferably streptavidin.

[0427] In still a further embodiment of the fourth detection method according to the invention, the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides are covalently linked to a spacer, which spacer is itself also covalently bound to the substrate in order to immobilise the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides onto said substrate. Such a spacer may be a peptide polymer such as a poly-alanine or a poly-lysine peptide of 10 to 15 amino acids in length.

[0428] In still a further embodiment of the fourth detection method above, the detection step c) consists of detecting changes in the optical characteristics of the substrate onto which the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides are bound.

[0429] In yet a further embodiment of the fourth detection method of the invention, the detection step c) consists of bringing into contact the substrate wherein complexes are formed between the targeted polypeptide molecule contained in the sample assayed and the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides bound to said support, with a detectable molecule having the ability to bind to such complexes.

[0430] A further object of the invention consists of a device or an apparatus for the detection of a polypeptide or a plurality of polypeptides of interest within a sample, wherein said device or apparatus comprises a substrate onto which a Selected Interacting Domain (SID®) polypeptide (or a plurality of Selected Interacting Domain (SID®) polypeptides) is (are) immobilised.

[0431] Such a device or apparatus of the invention above may comprise or consist of a suitable substrate onto which the plurality of Selected Interacting Domain (SID®) polypeptides are arranged in an ordered manner, thus forming an area such as described above.

Pharmaceutical Compositions Containing a Selected Interacting Domain (SID®) Polypeptide

[0432] It results from the method according to which a Selected Interacting Domain (SID®) polypeptide of SEQ ID N^(o)1 to 38 has been selected and characterized that such a Selected Interacting Domain (SID®) polypeptide or a variant thereof is both:

[0433] (i) endowed with highly specific binding properties to a (bait) polypeptide of interest; and

[0434] (ii) devoid ed of the biological activity of the naturally occurring protein from which this Selected Interacting Domain (SID®) polypeptide or a variant thereof is derived.

[0435] These original properties of a Selected Interacting Domain (SID®) polypeptide of SEQ ID N^(o)1 to 38 or a variant thereof allow its use for interfering with a naturally occurring interaction between a first protein and a second protein within the cell of an organism by the binding of said Selected Interacting Domain (SID®) polypeptide specifically either to said first polypeptide or said second polypeptide.

[0436] The (SID®) polypeptides of the invention or variants thereof are capable of interfering with the in vivo protein-protein interactions between HCV proteins or between a HCV protein and a protein from the organism which has been infected with the Hepatitis C virus.

[0437] For example the SID® polypeptide of SEQ ID N^(o)2 interferes with the naturally occurring interaction between the core and the NS3 protein HCV. Similarly, the SID® polypeptide of SEQ ID N^(o)17 interferes with the interaction between the NS4A and the NS4B proteins (see table

[0438] Thus, another object of the invention consists of a pharmaceutical composition comprising a pharmaceutically effective amount of a Selected Interacting Domain (SID®) polypeptide or a variant thereof.

[0439] The invention also relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a nucleic acid comprising a polynucleotide encoding a Selected Interacting Domain (SID®) polypeptide of SEQ ID N^(o)1 to 38 or a variant thereof which polynucleotide is placed under the control of an appropriate regulatory sequence.

[0440] Preferred nucleic acids are the nucleotide sequences SEQ ID N^(o)39 to 76.

[0441] The invention also pertains to a pharmaceutical composition comprising a pharmaceutically effective amount of a recombinant expression vector comprising a polynucleotide encoding the Selected Interacting Domain (SID®) polypeptide or a variant thereof.

[0442] The invention also pertains to a method for preventing or curing a viral infection by a hepatitis C virus in a human or an animal, wherein said method comprises a step of administering to the human or animal body a pharmaceutically effective amount of a Selected Interacting Domain (SID®) polypeptide of SEQ ID N^(o)1 to 38 or a variant thereof which binds to a targeted viral or mammal, typically- human protein.

[0443] A pharmaceutical composition as described above, wherein said composition is administered by any route, such as intravenous route, intramuscular route, oral route, or mucosa route with an acceptable physiological carrier and/or adjuvant, also forms part of the invention.

[0444] The Selected Interacting Domain (SID®) polypeptide or a variant thereof as a medicament for the prevention and/or treatment of pathologies induced by HCV are the most preferred.

[0445] The Selected Interacting Domain (SID®) polypeptides of SEQ ID N^(o)1 to 38 as active ingredients of a pharmaceutical composition will be preferably in a soluble form combined with a pharmaceutically acceptable vehicle.

[0446] Such compounds which can be used in a pharmaceutical composition offer a new approach for preventing and/or treating pathologies linked to infection by HCV. Preferably, these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular or intra dermal route or by the oral route.

[0447] Their modes of administration, optimum dosages and galvanic forms can be determined according to the criteria generally taken into account in establishing a treatment suited to a patient, such as for example the age or body weight of the patient, the seriousness of his general condition, the tolerance to treatment and the side effects observed, and the like.

[0448] The identified compound can be administered to a mammal, including a human patient, alone or in pharmaceutical compositions where they are mixed with suitable carriers or excipients at therapeutically effective doses to treat disorders associated with prokaryotic micro-organism infection. Techniques for formulation and administration of the compounds of the invention may be found in “Remington's Pharmaceutical Sciences” Mack Publication Co., Easton, Pa., latest edition.

[0449] For any Selected Interacting Domain (SID®) polypeptide or any variant thereof used according to the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown the desired effect in an in vitro system. Such information can be used to more accurately determine useful doses in humans.

[0450] A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50 Compounds which exhibit high therapeutic indices are preferred.

[0451] The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g. Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, CH.I).

[0452] Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain the modulating effects. Dosages necessary to achieve the modulating effect will depend on individual characteristics and route of administration.

[0453] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgement of the prescribing physician.

[0454] The invention also pertains to a method for preventing or curing a viral in a human or an animal, wherein said method comprises the step of administering to the human or animal body a pharmaceutically effective amount of a nucleic acid comprising a polynucleotide encoding a Selected Interacting Domain (SD®) polypeptide of SEQ ID N^(o)1 to 38, or a variant thereof, and wherein said polynucleotide is placed under the control of a regulatory sequence which is functional in said human or said animal.

[0455] Preferred polynucleotides are the nucleic acids of SEQ ID N^(o)39 to 76.

[0456] The invention also relates to a method for preventing or curing a viral or in a human or an animal, wherein said method comprises the step of administering to the human or animal body a pharmaceutically effective amount of a recombinant expression vector comprising a polynucleotide encoding a Selected Interacting Domain (SD®) polypeptide which binds to a viral or bacterial protein.

[0457] Other characteristics and advantages of the invention appear in the remainder of the description with the examples below, without linking the invention in any manner.

EXAMPLES

[0458] Preparation of a HCV Genomic Collection

[0459] 1.A. Collection Preparation and Transformation in Escherichia coli

[0460] 1.A.1 Fragmentation of genomic DNA preparation.

[0461] The genomic DNA of the infectious HCV strain H77 (Yanagi et al., P.N.A.S. 1997, 94, 8738-43) is fragmented in a nebulizer (GATC) for 2 minutes at a pressure of 2 bars, precipitated and resuspended in water.

[0462] The obtained nubilized genomic DNA is successively treated with Mung Bean Nuclease (Biolabs) (30 minutes at 30° C.), T4 DNA polymerase (Biolabs) (10 minutes at 37° C.) and Klenow enzyme (Pharmacia) (10 minutes at room temperature and 1 hour at 16° C.).

[0463] DNA is then extracted, precipitated and resuspended in water.

[0464] 1.A.2. Ligation of linkers to blunt-ended genomic DNA

[0465] Oligonucleotide HGX931 (5′ end phosphorylated) 1 μg/μl and HGX932 1 μg/μl.

[0466] Sequence of the oligo HGX931: 5′-GGGCCACGAA-3′ (SEQ ID N^(o)151). Sequence of the oligo HGX932: 5′-TTCGTGGCCCCTG-3′(SEQ ID N^(o)152).

[0467] Linkers were preincubated (5 minutes at 95° C., 10 minutes at 68° C., 15 minutes at 42° C.) then cooled down at room temperature and ligated with genomic DNA inserts at 16° C. overnight.

[0468] Linkers were further removed on a separation column (Chromaspin TE 400, Clontech), according to the manufacturer's protocol.

[0469] 1.A.3. Vector preparation

[0470] Plasmid pP6 (see FIG. 13) was prepared by replacing the Spe1/Xho1 fragment of pGAD3S2X with the double-stranded oligonucleotide: 5′CTAGCCATGGCCGCAGGGGCCGCGGCCGCACTAGTGGGGATCCTTAAT TAAAGGGCCACTGGGGCCCCCCGTACCGGCGTCCCCGGCGCCGGCGTGAT CACCCCTAGGAATTAATTTCCCGGTGACCCCGGGGGAGCT 3′ (SEQ ID N^(o) 153).

[0471] The pP6 vector is successively digested with Sfi1 and BamH1 restriction enzymes (Biolabs) for 1 hour at 37° C., extracted, precipitated and resuspended in water. Digested plasmid vector backbones are purified on a separation column (Chromaspin TE 400, Clontech), according to the manufacturer's protocol.

[0472] 1.A.4 Ligation between vector and insert of genomic DNA

[0473] The prepared vector is ligated overnight at 15° C. with the genomic blunt-ended DNA described in section 2 using T4 DNA ligase (Biolabs). The DNA is then precipitated and resuspended in water.

[0474] 1.A.5. Library transformation in Escherichia coli

[0475] Transform DNA from section 1.A.4. into Electromax DH10B electrocompetent ells (Gibco BRL) with Cell Porator apparatus (Gibco BRL). Add 1 ml SOC medium and incubate transformed cells at 37° C. for 1 hour. Add 9 ml volume of SOC medium per tube and plate on LB+ampicillin medium. Scrape colonies with liquid LB medium. Aliquot and freeze at −80° C.

[0476] The obtained collection of recombinant cell clones is named HGXBHCV1.

[0477] 1.B. Collection transformation in Saccharomyces cerevisiae

[0478] The Saccharomyces cerevisiae strain (Y187 (MATα Gal4Δ Ga180Δ ade2-101 His3 Leu2-3, −112 Trp1-901 Ura3-52 URA3::UASGAL1-LacZ Met) transformed with the HGXBHCV1 HCV genomic DNA library.

[0479] The plasmid DNA contained in E. coli are extracted (Qiagen) from aliquoted E. coli frozen cells (1.A.5.).

[0480] Grow Saccharomyces cerevisiae yeast Y187 in YPGlu.

[0481] Yeast transformation is performed according to standard protocol (GIEST et al. Yeast, 11, 355-360, 1995) using yeast carrier DNA (Clontech). This experiment leads to 10⁴ to 5.10⁴ cells/μg DNA. Spread 2.10⁴ cells on DO-Leu medium per plates. Aliquot and freeze at −80° C. The obtained collection of recombinant cell clones is named HGXYHCV1.

[0482] 1.C. Construction of bait plasmids

[0483] Plasmid pB5 (see FIG. 12) is prepared by replacing the NcoI/SalI polylinker fragment with the double-stranded oligonucleotide. 5′CATGGCCGCAGGGGCCGCGGCCGCACTAGTGGGGATCCTTAATTAAA GGGCCACTGGGGCCCCCCGGCGTCCCCGGCGCCGGCGTGATCACCCCTAG GAATTAATTTCCCGGTGACCCCGGGGGAGCT 3′. ( SEQ ID N^(o) 154).

[0484] The linkered genomic DNA described in section 2 is ligated into pB5 that has been digested with Sfi1 restriction enzyme and DNA transformed into competent E. coli Cells are grown and plasmid DNA extracted and sequenced. Those plasmids which code in-frame fusion proteins are used as bait plasmids.

EXAMPLE 2

[0485] Screening the Collection with the Two-hybrid in Yeast System

[0486] 2.A. The Mating Protocol

[0487] We have chosen the mating two-hybrid in yeast system (firstly described by FROMONT-RACINE et al., Nature Genetics, 1997, vol. 16, 277-282, Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens) for its advantages but we could also screen the HCV collection in classical two-hybrid system as described in Fields et al. or in a yeast reverse two-hybrid system.

[0488] The mating procedure allows a direct selection on selective plates because the two fusion proteins are already produced in the parental cells. No replica plating is required. This protocol is written for the use of the library transformed into the Y187 strain.

[0489] Before mating, transform S. cerevisiae (CG 1945 strain (MATa Ga14-542 Gal180-538 ade2-101 His3*200 Leu2-3, −112 Trp1-901 Ura3-52 Lys2-801 URA::GAL4 17 mers (X3)- CyC1TATA-LacZ LYS2::GAL1UAS-GAL1TATA-HIS3 CYH^(R))) according to step 1.B. and spread on DO-Trp medium.

[0490] Day 1, Morning: Preculture

[0491] Preculture of Y187 cells carrying the bait plasmid obtained at step 1.C. in 20 ml DO-Trp medium. Grow at 30° C. with vigorous agitation.

[0492] Day 1, Late Afternoon: Culture

[0493] Measure OD₆₀₀nm of the DO-Trp pre-culture of Y187 cells carrying the bait plasmid preculture. The OD_(600nm) must lie between 0.1 and 0.5 in order to correspond to a linear measurement.

[0494] Inoculate 50 ml DO-Trp at OD_(600nm) 0.006/ml, grow overnight at 30° C. with vigorous agitation.

[0495] Day 2: Mating

[0496] medium and plates

[0497] 1 YPGlu 15 cm plate

[0498] 50 ml tube with 13 ml DO-Leu-Trp-His

[0499] 100 ml flask with 5 ml of YPGlu

[0500] 30 8 DO-Leu-Trp-His plates

[0501] 2 DO-Leu plates

[0502] 2 DO-Trp plates

[0503] 2 DO-Leu-Trp plates

[0504] Measure OD_(600nm) of the DO-Trp culture. It should be around 1.

[0505] For the mating, you must use twice as many bait cells as library cells. To get a good mating efficiency, you must collect the cells at 10⁸ cells per cm².

[0506] Estimate the amount of bait culture (in ml) that makes up 30 OD_(600nm) units for the mating with the prey library.

[0507] Thaw a vial containing the HGXYHCV1 library slowly on ice. Add the 0.5 ml of the vial to 5 ml YPGlu. Let those cells recover at 30° C., under gentle agitation for 10 minutes.

[0508] Mating

[0509] Put the 30 OD_(600nm) units of bait culture into a 50 ml flacon tube.

[0510] Add the HGXYHCV1 library culture to the bait culture. Centrifuge, discard the supernatant and resuspend in 0.8 ml YPGlu medium.

[0511] Distribute the cells onto a YPGlu plate with glass beads. Spread cells by shaking the plates.

[0512] Incubate the plate cells-up at 30° C. for 4 h 30 min.

[0513] Collection of Mated Cells

[0514] Wash and rinse the plate with 6 ml and 7 ml consecutively of DO-Leu-Trp-His.

[0515] Perform two parallel serial ten-fold dilutions in 500 μl DO-Leu-Trp-His up to 1/10,000. Spread out 50 μl of each 1/10000 dilution onto DO-Leu and DO-trp plates and 50 μl of each 1/1000 dilution onto DO-Leu-Trp plates.

[0516] Spread 3.2 ml of collected cells in 400 μl aliquots on DO-Leu-Trp-His+Tet plates.

[0517] DAY 4

[0518] Selection of clones able to grow on DO-Leu-Trp-His+Tetracyclin: this medium allows us to isolate diploid clones presenting an interaction.

[0519] Count the Trp+Leu+ colonies on control plates and the total number of His+ colonies on the DO-Leu-Trp-His+Tetracyclin plates.

[0520] The number of His+ cell clones will define which protocol is to be processed:

[0521] Upon 2.10⁶ Trp+Leu+ colonies:

[0522] if number of His+ cell clones <95: then process luminometry protocol on all colonies;

[0523] if number of His+ cell clones >95 and <5000: then process luminometry protocol on 95 colonies;

[0524] if number of His+ cell clones >500: repeat screen using DO-Leu-Trp-His+Tetracyclin plates containing 3-aminotriazol.

[0525] 2.B The Luminometry Assay

[0526] Grow His+colonies overnight at 30° C. in microtiter plates containing DO-Leu-Trp-His-Tetracyclin medium with shaking. The day after, dilute 15 times overnight culture into a new microtiter plate containing the same medium. Incubate 5 hours at 30° C. with shaking. Dilute samples 5 times and read OD_(600nm). Dilute again to obtain between 10 000 and 75 000 yeast cells/well in 100 μl final volume.

[0527] Per well, add 76 μl of One Step Yeast Lysis Buffer (Tropix), 20 μl Sapphirell Enhancer (Tropix), 4 μl Galacton Star (Tropix), incubate 40 minutes at 30° C.

[0528] Measure the β-Gal read-out (L) using a Luminometer (Trilux, Wallach).

[0529] Calculate value of OD_(600nm)×L and selected interacting preys having highest values.

[0530] At this step of the protocol, we have isolated diploid cell clones presenting interaction. The next step is now to identify polypeptides involved in the selected interactions.

EXAMPLE 3

[0531] Identification of Positive Clones

[0532] 3.A. PCR on Yeast Colonies

[0533] Introduction

[0534] PCR amplification of fragments of plasmid DNA directly on yeast colonies is a quick and efficient procedure to identify sequences cloned into this plasmid. It is directly derived from a published protocol (Wang H. et al., Analytical Biochemistry, 237, 145-146, 1996). However, it is not a standardized protocol: in our hands it varies from strain to strain, and is dependent on experimental conditions (number of cells, Taq polymerase source, etc). This protocol should be optimized to specific local conditions.

[0535] Materials

[0536] For 1 well, PCR mix composition is:

[0537] 32.5 μl water,

[0538] 5 μl 10× PCR buffer (Pharmacia),

[0539] 1 μl dNTP 10 mM,      0.5 μl Taq polymerase (85 μ/μl -Pharmacia),      0.5   μl  oligonucleotide   ABS1   10   pmole/μl:5′- GCGTTTGGAATCACTACAGG-3′,      0.5   μl  oligonucleotide   ABS2   10   pmole/μl:5′- CACGATGCACGTTGAAGTG-3′. - 1N NaOH.

[0540] Experiment

[0541] Grow positive colonies overnight at 30° C. on a 96 well cell culture cluster (Costar), containing 150 μl DO-Leu-Trp-His+Tetracyclin with shaking. Resuspend culture and transfer immediately 100 μl on a Thermowell 96 (Costar).

[0542] Centrifuge 5 minutes at 4000 rpm at room temperature.

[0543] Remove supernatant. Dispense 5 μl NaOH in each well, shake 1 minute.

[0544] Place the Thermowell in the thermocycler (GeneAmp 9700, Perkin Elmer) 5 minutes at 99,9° C. and then 10 minutes at 4° C.

[0545] In each well, add PCR mix, shake well.

[0546] Set up the PCR program as followed: 94° C. 3 minutes 94° C. 30 seconds 53° C. 1 minute 30 seconds ×35 cycles 72° C. 3 minutes 72° C. 5 minutes 15° C. ∞

[0547] Check the quality, the quantity and the length of the PCR fragment on agarose gel.

[0548] The length of the cloned fragment is the estimated length of the PCR fragment minus 300 base pairs that correspond to the amplified flanking plasmid sequences.

[0549] 3.B Plasmids Rescue from Yeast by Electroporation

[0550] Introduction

[0551] The previous protocol of PCR on yeast cell may not be successful, in such a case, we rescue plasmids from yeast by electroporation. This experiment allows the recovery of prey plasmids from yeast cells by transformation of E.coli with a yeast cellular extract.

[0552] We can then amplify the prey plasmid and sequence the cloned fragment.

[0553] Material

[0554] Plasmid Rescue

[0555] Glass beads 425-600 μm (Sigma)

[0556] Phenol/chloroform (1/1) premixed with isoamyl alcohol (Amresco)

[0557] Extraction buffer: 2% Triton ×100, 1% SDS, 100 mM NaCl, 10 mM TrisHCl pH 8,0, 1 mM EDTA pH 8.0.

[0558] Mix ethanol/NH₄Ac: 6 volumes ethanol with 7.5 M NH₄ Acetate, 70% Ethanol and yeast cells in patches on plates.

[0559] Electroporation

[0560] SOC medium

[0561] M9 medium

[0562] Selective plates: M9-Leu+Ampicillin

[0563] 2 mm electroporation cuvettes (Eurogentec)

[0564] Experiment

[0565] Plasmid Rescue

[0566] Prepare cell patch on DO-Leu-Trp-His with cell culture of section 2.C.

[0567] Scrape the cell of each patch in Eppendorf tube, add 300 μl of glass beads in each tube, then add 200 μl extraction buffer and add 200 μl phenol: chloroform:isoamyl alcohol (25:24:1).

[0568] Centrifuge tubes 10 minutes at 15000 rpm.

[0569] Transfer 180 μl supernatant to a sterile Eppendorf tube and add to each 500 μl ethanol/NH₄Ac, vortex.

[0570] Centrifuge tubes 15 minutes, 15000 rmp at 4° C.

[0571] Wash pellet with 200 μl 70% ethanol, remove ethanol and dry pellet, Resuspend pellet in 10 μl water. Store extracts at −20° C.

[0572] Electroporation

[0573] Material: Electrocompetent MC1066 cells prepared according to standard protocols (Maniatis).

[0574] Add 1 μl of yeast plasmid DNA-extract to pre-chilled Eppendorf tube, and keep on ice.

[0575] Mix 1 μl plasmid yeast DNA-extract sample, add 20 μl electrocompetent cells and transfer in a cold electroporation cuvette.

[0576] Set the Biorad electroporator on 200 ohms resistance, 25 μF capacity; 2.5 kV. Place cuvette in the cuvette holder and electroporate.

[0577] Add 1 ml SOC into the cuvette and transfer the cell-mix into sterile Eppendorf tube.

[0578] Let cells recover for 30 minutes at 37° C., spin the cells down 1 minute, 4000× g and pour off supernatant. Keep about 100 μl medium and use it to resuspend the cells and spread them on selective plates (e.g. M9-Leu plates).

[0579] Incubate plates for 36 hours at 37° C.

[0580] Grow one colony and extract plasmids. Check presence and size of insert through enzymatic digestion and agarose gel. Sequence insert.

EXAMPLE 4

[0581] Protein-Protein Interaction

[0582] For each bait, the previously protocol leads to the identification of prey polynucleotide sequences. Using a suitable software program (eg Blastwun, available on the Internel site of the University of Washington: http:/bioweb.pasteur.fr/seqanal/interfaces/blastwu.html) the region of the HCV genome is encoded by the prey fragment may be determined and whether the fusion proteins encoded are in the same open reading frame of translation as the HCV polyprotein or not.

EXAMPLE 5

[0583] Identification of SID®

[0584] The presence of contiguous polypeptides in the HCV genome and the high complexity of the prey library used prevents the determination of SID®s by previous means since prey fragments can overlap multiple polypeptides. The high complexity of the prey library used relative to the small genome size also prevented such a simple analysis since prey fragments can overlap multiple interacting domains. It was also necessary to overcome the problems caused by protein preys encoded by out-of-frame fusions of regions of the HCV genome.

[0585] In order to determine the SID®s for a particular bait protein, it was therefore necessary to devise a suitable algorithm which would take into account all these problems:

[0586] 5.1. The prey fragments are initially sorted according to which reading frame of the polypeptide sequence they correspond to. This enables the separation of physiologically relevant prey protein from out-of-frame fusions which bind in the two-hybrid assay.

[0587] 5.2. Each prey fragment is compared pairwise with other prey fragments and two fragments are clustered together if they overlap by more than 30% of their lengths (see FIG. 8). Further fragments are assigned to the cluster if, and only if, overlap all the fragments in the cluster by more than 30% of their length.

[0588] 5.3 For each cluster of fragments thus produced, a pre-SID is defined as the intersection of all the fragments present in the cluster defined in 5.2 (FIG. 9).

[0589] 5.4. The pre-SIDs defined in 5.3 are then analysed pairwise and if the region of intersection between two pre-SIDs is greater than 30 bp then a SID® is defined as this region of intersection. If the non-intersecting region of a pre-SID is of more than 30 bp in length and this non-intersecting region represents more than 30% of the length of one of the fragments that comprises this region, then this non-intersecting region is also defined as a SID®s (FIG. 10).

[0590] 5.5 The number of fragments contributing to each SID defined in 5.4 is counted. In the case of overlapping SIDs®, the SID® which contains the most fragments is identified, and all the fragments which contribute to this SID® are removed from overlapping SIDs®. The inspection of the fragments which remain in these overlapping SIDs® determines the final sequence of the SID® (FIG. 11). TABLE 1 Summary of the protein-proptein interactions between the SID polypeptides of the invention and H77 strain HCV polypeptides SEQ SEQ SEQ SEQ ID ID begin end ID ID N^(o) begin end N^(o) Bait N^(o) (1) (2) (2) N^(o) (3) SID (4) (2) (2) (5) Core (87%) 114  302  614 77 Core (100%) 39  446  600  1 Core (100%) 115  342  683 78 NS3 (100%) 40 4814 4922  2 Core (100%) 115  342  683 78 Core (100%) 41  380  616  3 E1 (100%) 116  995 1342 79 E2 (100%) 42 1871 1987  4 E1 (4%)/E2 117 1478 1756 80 NS3 (100%) 43 4787 5242  5 (95%) E2 (100%) 118 1745 2278 81 E2 (100%) 44 1871 1958  6 E2 (100%) 119 1799 2090 82 E2 (100%) 45 1808 1890  8 NS2 (12%)/NS3 120 3312 4150 83 NS4A (59%)/ 46 5375 5542  9 (87%) NS4B (40%) NS3 (100%) 121 3767 4244 84 NS3 (100%) 47 4676 4801 10 NS3 (100%) 122 3779 4571 85 NS3 (100%) 48 4856 4945 11 NS3 (100%) 123 3974 4559 86 NS3 (100%) 49 4817 4903 12 NS3 (100%) 124 4238 4857 87 NS5B (100%) 50 7979 8109 13 NS3 (100%) 125 4298 4859 88 NS3 (100%) 51 4031 4118 14 NS3 (100%) 126 4691 5168 89 E2 (100%) 52 1784 1888 15 NS3 (100%) 127 4838 5230 90 E2 (100%) 53 1871 1968 16 NS3 (1%)/NS4 128 5310 5467 91 NS4B (100%) 54 5918 6154 17 A (98%) NS4A (100%) 129 5342 5400 92 NS3 (100%) 55 3512 3956 18 NS4B (86%)/N 130 5717 6344 93 NS4B (53%)/ 56 6197 6310 19 S5A (13%) NS5A (46%) NS4B (70%)/N 131 5819 6444 94 E2 (100%) 57 1844 1933 20 S5A (29%) NS4B (55%)/N 132 5882 6562 95 NS5B (100%) 58 9083 9222 21 S5A (44%) NS4B (82%)/N 133 5897 6335 96 NS4B (100%) 59 5819 6080 22 S5A(17%) NS4B (100%) 134 6011 6177 97 E2 (100%) 60 1823 1955 23 NS4B (30%)/N 135 6107 6605 98 NS4B (100%) 61 5879 6072 24 S5A (69%) NS4B (12%)/N 136 6141 7069 99 E2 (100%) 62 1784 1875 25 S5A (87%) NS4B (8%)/NS 137 6182 7034 100  E1 (100%) 63 1226 1458 26 5A (91%) NS4B (9%)/NS 138 6188 6939 101  NS4B (70%)/ 64 6176 6291 27 5A (90%) NS5A (28%) NS5A (100%) 139 6317 6576 102  NS3 (100%) 65 4784 4928 28 NS5A (100%) 140 6440 6727 103  NS5A (100%) 66 6557 6721 29 NS5A (100%) 141 7019 7249 104  NS3 (100%) 67 4451 4790 30 NS5A (100%) 142 7274 7549 105  NS4B (100%) 68 6029 6194 31 NS5B (100%) 143 7613 8027 106  NS5B (100%) 69 8354 8665 32 NS5B (100%) 144 7838 8743 107  NS5B (100%) 70 7769 8011 33 NS5B (100%) 145 7856 8458 108  NS3 (100%) 71 4715 4901 34 NS5B (100%) 146 7976 8759 109  NS5B (100%) 72 7775 8011 35 NS5B (100%) 147 8564 8948 110  E2 (100%) 73 1805 1887 36 NS5B (100%) 148 8708 8978 111  E2 (100%) 74 1751 1865 37 NS5B (100%) 149 8996 9220 112  NS4B (57%)/ 75 6194 6303 38 NS5A(41%) NS5B (100%) 150 9032 9226 113  NS4B(63%)/ 76 6206 6286 39 NS5A(35%)

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1 156 1 50 PRT Hepatitis C virus 1 Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys 1 5 10 15 Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys 20 25 30 Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp 35 40 45 Pro Leu 50 2 35 PRT Hepatitis C virus 2 Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg 1 5 10 15 Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala 20 25 30 Gly Cys Ala 35 3 77 PRT Hepatitis C virus 3 Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln 1 5 10 15 Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly 20 25 30 Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg 35 40 45 Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala 50 55 60 Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly 65 70 75 4 37 PRT Hepatitis C virus 4 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr 1 5 10 15 Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr 20 25 30 Arg Pro Pro Leu Gly 35 5 150 PRT Hepatitis C virus 5 Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile 1 5 10 15 Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser 20 25 30 Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu 35 40 45 Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro 50 55 60 Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe 65 70 75 80 Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln 85 90 95 Ser Gly Glu Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys 100 105 110 Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys 115 120 125 Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr 130 135 140 Arg Leu Gly Ala Val Gln 145 150 6 28 PRT Hepatitis C virus 6 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr 1 5 10 15 Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val 20 25 7 26 PRT Hepatitis C virus 7 Pro Pro Arg Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro 1 5 10 15 Val Tyr Cys Phe Thr Pro Ser Pro Val Val 20 25 8 54 PRT Hepatitis C virus 8 Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile 1 5 10 15 Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu 20 25 30 Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu 35 40 45 Gln Phe Lys Gln Lys Ala 50 9 40 PRT Hepatitis C virus 9 Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr 1 5 10 15 Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Leu 20 25 30 Pro Gln Asp Ala Val Ser Arg Thr 35 40 10 28 PRT Hepatitis C virus 10 Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr 1 5 10 15 Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala 20 25 11 27 PRT Hepatitis C virus 11 Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro 1 5 10 15 Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu 20 25 12 42 PRT Hepatitis C virus 12 Leu Glu Asp Ser Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn 1 5 10 15 Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg 20 25 30 Leu Ile Val Phe Pro Asp Leu Gly Val Arg 35 40 13 28 PRT Hepatitis C virus 13 Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala 1 5 10 15 Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val 20 25 14 33 PRT Hepatitis C virus 14 Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val 1 5 10 15 Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro 20 25 30 Val 15 31 PRT Hepatitis C virus 15 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr 1 5 10 15 Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn 20 25 30 16 77 PRT Hepatitis C virus 16 Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser 1 5 10 15 Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala 20 25 30 Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly 35 40 45 Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala 50 55 60 Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro 65 70 75 17 147 PRT Hepatitis C virus 17 Glu Val Gln Ile Val Ser Thr Ala Thr Gln Thr Phe Leu Ala Thr Cys 1 5 10 15 Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr 20 25 30 Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp 35 40 45 Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser Arg Ser Leu Thr 50 55 60 Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala 65 70 75 80 Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu 85 90 95 Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu 100 105 110 Leu Cys Pro Ala Gly His Ala Val Gly Leu Phe Arg Ala Ala Val Cys 115 120 125 Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Asn Leu 130 135 140 Gly Thr Thr 145 18 36 PRT Hepatitis C virus 18 Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys 1 5 10 15 Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile 20 25 30 Cys Glu Val Leu 35 19 28 PRT Hepatitis C virus 19 Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly 1 5 10 15 Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp 20 25 20 45 PRT Hepatitis C virus 20 Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg Ala Arg 1 5 10 15 Leu Leu Ser Arg Gly Gly Arg Ala Ala Ile Cys Gly Lys Tyr Leu Phe 20 25 30 Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile 35 40 45 21 86 PRT Hepatitis C virus 21 Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly Ser Val 1 5 10 15 Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly 20 25 30 Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro 35 40 45 Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly 50 55 60 Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 65 70 75 80 Gly Pro Gly Glu Gly Ala 85 22 43 PRT Hepatitis C virus 22 Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr 1 5 10 15 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr 20 25 30 Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe 35 40 23 63 PRT Hepatitis C virus 23 Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala 1 5 10 15 Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp 20 25 30 Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val 35 40 45 Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly 50 55 60 24 29 PRT Hepatitis C virus 24 Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val 1 5 10 15 Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr 20 25 25 76 PRT Hepatitis C virus 25 Arg Arg His Trp Thr Thr Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly 1 5 10 15 His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser 20 25 30 Pro Thr Ala Ala Leu Val Val Ala Gln Leu Leu Arg Ile Pro Gln Ala 35 40 45 Ile Met Asp Met Ile Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile 50 55 60 Ala Tyr Phe Ser Met Val Gly Asn Trp Ala Lys Val 65 70 75 26 37 PRT Hepatitis C virus 26 Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His 1 5 10 15 Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu 20 25 30 Arg Asp Ile Trp Asp 35 27 47 PRT Hepatitis C virus 27 Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly 1 5 10 15 Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp 20 25 30 Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr 35 40 45 28 53 PRT Hepatitis C virus 28 Leu Pro Ala Pro Asn Tyr Lys Phe Ala Leu Trp Arg Val Ser Ala Glu 1 5 10 15 Glu Tyr Val Glu Ile Arg Arg Val Gly Asp Phe His Tyr Val Ser Gly 20 25 30 Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Ile Pro Ser Pro Glu 35 40 45 Phe Phe Thr Glu Leu 50 29 112 PRT Hepatitis C virus 29 Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys 1 5 10 15 Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu 20 25 30 Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr 35 40 45 Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val 50 55 60 Val Ser Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser 65 70 75 80 Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu 85 90 95 Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gln Asp Ala Val 100 105 110 30 54 PRT Hepatitis C virus 30 Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala 1 5 10 15 Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His 20 25 30 Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val 35 40 45 Thr Ala Ile Leu Ser Ser 50 31 102 PRT Hepatitis C virus 31 Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr 1 5 10 15 Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly 20 25 30 Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Ile Lys Ala 35 40 45 Arg Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys Thr Met Leu Val 50 55 60 Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu 65 70 75 80 Asp Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser 85 90 95 Ala Pro Pro Gly Asp Pro 100 32 79 PRT Hepatitis C virus 32 Leu Gln Val Leu Asp Ser His Tyr Gln Asp Val Leu Lys Glu Val Lys 1 5 10 15 Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala 20 25 30 Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly 35 40 45 Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Ala His Ile Asn 50 55 60 Ser Val Trp Lys Asp Leu Leu Glu Asp Ser Val Thr Pro Ile Asp 65 70 75 33 61 PRT Hepatitis C virus 33 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr 1 5 10 15 Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg 20 25 30 Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu 35 40 45 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu 50 55 60 34 77 PRT Hepatitis C virus 34 Val Leu Asp Ser His Tyr Gln Asp Val Leu Lys Glu Val Lys Ala Ala 1 5 10 15 Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser 20 25 30 Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys 35 40 45 Asp Val Arg Cys His Ala Arg Lys Ala Val Ala His Ile Asn Ser Val 50 55 60 Trp Lys Asp Leu Leu Glu Asp Ser Val Thr Pro Ile Asp 65 70 75 35 26 PRT Hepatitis C virus 35 Tyr Pro Pro Arg Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly 1 5 10 15 Pro Val Tyr Cys Phe Thr Pro Ser Pro Val 20 25 36 37 PRT Hepatitis C virus 36 Pro Ile Ser Tyr Ala Asn Gly Ser Gly Leu Asp Glu Arg Pro Tyr Cys 1 5 10 15 Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val Pro Ala Lys Ser Val 20 25 30 Cys Gly Pro Val Tyr 35 37 35 PRT Hepatitis C virus 37 Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu 1 5 10 15 Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp 20 25 30 Ile Cys Glu 35 38 25 PRT Hepatitis C virus 38 Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro 1 5 10 15 Cys Ser Gly Ser Trp Leu Arg Asp Ile 20 25 39 152 DNA Hepatitis C virus 39 cttgttgccg cgcaggggcc ctagattggg tgtgcgcgcg acgaggaaga cttccgagcg 60 gtcgcaacct cgaggtagac gtcagcctat ccccaaggca cgtcggcccg agggcaggac 120 ctgggctcag cccgggtacc cttggcccct ct 152 40 106 DNA Hepatitis C virus 40 tggcaggggg aagccaggca tctatagatt tgtggcaccg ggggagcgcc cctccggcat 60 gttcgactcg tccgtcctct gtgagtgcta tgacgcgggc tgtgct 106 41 234 DNA Hepatitis C virus 41 taacaccaac cgtcgcccac aggacgtcaa gttcccgggt ggcggtcaga tcgttggtgg 60 agtttacttg ttgccgcgca ggggccctag attgggtgtg cgcgcgacga ggaagacttc 120 cgagcggtcg caacctcgag gtagacgtca gcctatcccc aaggcacgtc ggcccgaggg 180 caggacctgg gctcagcccg ggtacccttg gcccctctat ggcaatgagg gttg 234 42 114 DNA Hepatitis C virus 42 tcccagcccc gtggtggtgg gaacgaccga caggtcgggc gcgcctacct acagctgggg 60 tgcaaatgat acggatgtct tcgtccttaa caacaccagg ccaccgctgg gcaa 114 43 453 DNA Hepatitis C virus 43 ctccaggact caacgccggg gcaggactgg cagggggaag ccaggcatct atagatttgt 60 ggcaccgggg gagcgcccct ccggcatgtt cgactcgtcc gtcctctgtg agtgctatga 120 cgcgggctgt gcttggtatg agctcacgcc cgccgagact acagttaggc tacgagcgta 180 catgaacacc ccggggcttc ccgtgtgcca ggaccatctt gaattttggg agggcgtctt 240 tacgggcctc actcatatag atgcccactt tttatcccag acaaagcaga gtggggagaa 300 ctttccttac ctggtagcgt accaagccac cgtgtgcgct agggctcaag cccctccccc 360 atcgtgggac cagatgtgga agtgtttgat ccgccttaaa cccaccctcc atgggccaac 420 acccctgcta tacagactgg gcgctgttca gaa 453 44 85 DNA Hepatitis C virus 44 tcccagcccc gtggtggtgg gaacgaccga caggtcgggc gcgcctacct acagctgggg 60 tgcaaatgat acggatgtct tcgtc 85 45 80 DNA Hepatitis C virus 45 ccctccaaga ccttgtggca ttgtgcccgc aaagagcgtg tgtggcccgg tatattgctt 60 cactcccagc cccgtggtgg 80 46 165 DNA Hepatitis C virus 46 ctgcgtggtc atagtgggca ggatcgtctt gtccgggaag ccggcaatta tacctgacag 60 ggaggttctc taccaggagt tcgatgagat ggaagagtgc tctcagcact taccgtacat 120 cgagcaaggg atgatgctcg ctgagcagtt caagcagaag gccct 165 47 123 DNA Hepatitis C virus 47 cggcgacttc gactctgtga tagactgcaa cacgtgtgtc actcagacag tcgatttcag 60 ccttgaccct acctttacca ttgagacaac cacgctcccc caggatgctg tctccaggac 120 tca 123 48 87 DNA Hepatitis C virus 48 ggagcgcccc tccggcatgt tcgactcgtc cgtcctctgt gagtgctatg acgcgggctg 60 tgcttggtat gagctcacgc ccgccga 87 49 84 DNA Hepatitis C virus 49 cagggggaag ccaggcatct atagatttgt ggcaccgggg gagcgcccct ccggcatgtt 60 cgactcgtcc gtcctctgtg agtg 84 50 128 DNA Hepatitis C virus 50 tctggaagac agtgtaacac caatagacac taccatcatg gccaagaacg aggttttctg 60 cgttcagcct gagaaggggg gtcgtaagcc agctcgtctc atcgtgttcc ccgacctggg 120 cgtgcgcg 128 51 85 DNA Hepatitis C virus 51 tcccaccggc agcggtaaga gcaccaaggt cccggctgcg tacgcagccc agggctacaa 60 ggtgttggtg ctcaacccct ctgtt 85 52 102 DNA Hepatitis C virus 52 cgaacgcccc tactgctggc actaccctcc aagaccttgt ggcattgtgc ccgcaaagag 60 cgtgtgtggc ccggtatatt gcttcactcc cagccccgtg gt 102 53 95 DNA Hepatitis C virus 53 tcccagcccc gtggtggtgg gaacgaccga caggtcgggc gcgcctacct acagctgggg 60 tgcaaatgat acggatgtct tcgtccttaa caaca 95 54 234 DNA Hepatitis C virus 54 ggcgggagct cttgtagcat tcaagatcat gagcggtgag gtcccctcca cggaggacct 60 ggtcaatctg ctgcccgcca tcctctcgcc tggagccctt gtagtcggtg tggtctgcgc 120 agcaatactg cgccggcacg ttggcccggg cgagggggca gtgcaatgga tgaaccggct 180 aatagccttc gcctcccggg ggaaccatgt ttcccccacg cactacgtgc cgga 234 55 442 DNA Hepatitis C virus 55 tgaggtccag atcgtgtcaa ctgctaccca aaccttcctg gcaacgtgca tcaatggggt 60 atgctggact gtctaccacg gggccggaac gaggaccatc gcatcaccca agggtcctgt 120 catccagatg tataccaatg tggaccaaga ccttgtgggc tggcccgctc ctcaaggttc 180 ccgctcattg acaccctgta cctgcggctc ctcggacctt tacctggtca cgaggcacgc 240 cgatgtcatt cccgtgcgcc ggcgaggtga tagcaggggt agcctgcttt cgccccggcc 300 catttcctac ttgaaaggct cctcgggggg tccgctgttg tgccccgcgg gacacgccgt 360 gggcctattc agggccgcgg tgtgcacccg tggagtggct aaagcggtgg actttatccc 420 tgtggagaac ctagggacaa cc 442 56 111 DNA Hepatitis C virus 56 tgtaacccag ctcctgaggc gactgcatca gtggataagc tcggagtgta ccactccatg 60 ctccggttcc tggctaaggg acatctggga ctggatatgc gaggtgctga g 111 57 87 DNA Hepatitis C virus 57 cgtgtgtggc ccggtatatt gcttcactcc cagccccgtg gtggtgggaa cgaccgacag 60 tcgggcgcg cctacctaca gctgggg 87 58 137 DNA Hepatitis C virus 58 cccgcccttg cgagcttgga gacaccgggc ccggagcgtc cgcgctaggc ttctgtccag 60 aggaggcagg gctgccatat gtggcaagta cctcttcaac tgggcagtaa gaacaaagct 120 caaactcact ccaatag 137 59 259 DNA Hepatitis C virus 59 tactgccttt gtgggtgctg gcctagctgg cgccgccatc ggcagcgttg gactggggaa 60 ggtcctcgtg gacattcttg cagggtatgg cgcgggcgtg gcgggagctc ttgtagcatt 120 caagatcatg agcggtgagg tcccctccac ggaggacctg gtcaatctgc tgcccgccat 180 cctctcgcct ggagcccttg tagtcggtgt ggtctgcgca gcaatactgc gccggcacgt 240 tggcccgggc gagggggca 259 60 130 DNA Hepatitis C virus 60 tggcattgtg cccgcaaaga gcgtgtgtgg cccggtatat tgcttcactc ccagccccgt 60 ggtggtggga acgaccgaca ggtcgggcgc gcctacctac agctggggtg caaatgatac 120 ggatgtcttc 130 61 191 DNA Hepatitis C virus 61 ggtcctcgtg gacattcttg cagggtatgg cgcgggcgtg gcgggagctc ttgtagcatt 60 caagatcatg agcggtgagg tcccctccac ggaggacctg gtcaatctgc tgcccgccat 120 cctctcgcct ggagcccttg tagtcggtgt ggtctgcgca gcaatactgc gccggcacgt 180 tggcccgggc g 191 62 89 DNA Hepatitis C virus 62 cgaacgcccc tactgctggc actaccctcc aagaccttgt ggcattgtgc ccgcaaagag 60 cgtgtgtggc ccggtatatt gcttcactc 89 63 230 DNA Hepatitis C virus 63 caggcgccac tggacgacgc aagactgcaa ttgttctatc tatcccggcc atataacggg 60 tcatcgcatg gcatgggata tgatgatgaa ctggtcccct acggcagcgt tggtggtagc 120 tcagctgctc cggatcccac aagccatcat ggacatgatc gctggtgctc actggggagt 180 cctggcgggc atagcgtatt tctccatggt ggggaactgg gcgaaggtcc 230 64 113 DNA Hepatitis C virus 64 tgccatactc agcagcctca ctgtaaccca gctcctgagg cgactgcatc agtggataag 60 ctcggagtgt accactccat gctccggttc ctggctaagg gacatctggg act 113 65 142 DNA Hepatitis C virus 65 tgtctccagg actcaacgcc ggggcaggac tggcaggggg aagccaggca tctatagatt 60 tgtggcaccg ggggagcgcc cctccggcat gttcgactcg tccgtcctct gtgagtgcta 120 tgacgcgggc tgtgcttggt at 142 66 162 DNA Hepatitis C virus 66 ccttcctgcg ccgaactata agttcgcgct gtggagggtg tctgcagagg aatacgtgga 60 gataaggcgg gtgggggact tccactacgt atcgggtatg actactgaca atcttaaatg 120 cccgtgccag atcccatcgc ccgaattttt cacagaattg ga 162 67 337 DNA Hepatitis C virus 67 cggagagatc cccttttacg gcaaggctat ccccctcgag gtgatcaagg ggggaagaca 60 tctcatcttc tgccactcaa agaagaagtg cgacgagctc gccgcgaagc tggtcgcatt 120 gggcatcaat gccgtggcct actaccgcgg tcttgacgtg tctgtcatcc cgaccagcgg 180 cgatgttgtc gtcgtgtcga ccgatgctct catgactggc tttaccggcg acttcgactc 240 tgtgatagac tgcaacacgt gtgtcactca gacagtcgat ttcagccttg accctacctt 300 taccattgag acaaccacgc tcccccagga tgctgtc 337 68 163 DNA Hepatitis C virus 68 ggtctgcgca gcaatactgc gccggcacgt tggcccgggc gagggggcag tgcaatggat 60 gaaccggcta atagccttcg cctcccgggg gaaccatgtt tcccccacgc actacgtgcc 120 ggagagcgat gcagccgccc gcgtcactgc catactcagc agc 163 69 309 DNA Hepatitis C virus 69 ggccatcaag tccctcactg agaggcttta tgttgggggc cctcttacca attcaagggg 60 ggaaaactgc ggctaccgca ggtgccgcgc gagcggcgta ctgacaacta gctgtggtaa 120 caccctcact tgctacatca aggcccgggc agcctgtcga gccgcagggc tccaggactg 180 caccatgctc gtgtgtggcg acgacttagt cgttatctgt gaaagtgcgg gggtccagga 240 ggacgcggcg agcctgagag ccttcacgga ggctatgacc aggtactccg ccccccccgg 300 ggacccccc 309 70 240 DNA Hepatitis C virus 70 actgcaagtt ctggacagcc attaccagga cgtgctcaag gaggtcaaag cagcggcgtc 60 aaaagtgaag gctaacttgc tatccgtaga ggaagcttgc agcctgacgc ccccacattc 120 agccaaatcc aagtttggct atggggcaaa agacgtccgt tgccatgcca gaaaggccgt 180 agcccacatc aactccgtgt ggaaagacct tctggaagac agtgtaacac caatagacac 240 71 184 DNA Hepatitis C virus 71 cactcagaca gtcgatttca gccttgaccc tacctttacc attgagacaa ccacgctccc 60 ccaggatgct gtctccagga ctcaacgccg gggcaggact ggcaggggga agccaggcat 120 ctatagattt gtggcaccgg gggagcgccc ctccggcatg ttcgactcgt ccgtcctctg 180 tgag 184 72 234 DNA Hepatitis C virus 72 agttctggac agccattacc aggacgtgct caaggaggtc aaagcagcgg cgtcaaaagt 60 gaaggctaac ttgctatccg tagaggaagc ttgcagcctg acgcccccac attcagccaa 120 atccaagttt ggctatgggg caaaagacgt ccgttgccat gccagaaagg ccgtagccca 180 catcaactcc gtgtggaaag accttctgga agacagtgta acaccaatag acac 234 73 80 DNA Hepatitis C virus 73 ctaccctcca agaccttgtg gcattgtgcc cgcaaagagc gtgtgtggcc cggtatattg 60 cttcactccc agccccgtgg 80 74 112 DNA Hepatitis C virus 74 tcctatcagt tatgccaacg gaagcggcct cgacgaacgc ccctactgct ggcactaccc 60 tccaagacct tgtggcattg tgcccgcaaa gagcgtgtgt ggcccggtat at 112 75 107 DNA Hepatitis C virus 75 cactgtaacc cagctcctga ggcgactgca tcagtggata agctcggagt gtaccactcc 60 atgctccggt tcctggctaa gggacatctg ggactggata tgcgagg 107 76 78 DNA Hepatitis C virus 76 gctcctgagg cgactgcatc agtggataag ctcggagtgt accactccat gctccggttc 60 ctggctaagg gacatctg 78 77 103 PRT Hepatitis C virus 77 Ala Cys Glu Cys Pro Gly Arg Ser Arg Arg Pro Cys Thr Met Ser Thr 1 5 10 15 Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro 20 25 30 Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr 35 40 45 Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys 50 55 60 Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys 65 70 75 80 Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp 85 90 95 Pro Leu Tyr Gly Asn Glu Gly 100 78 113 PRT Hepatitis C virus 78 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly 65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg 79 114 PRT Hepatitis C virus 79 Ala Ile Leu His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn 1 5 10 15 Ala Ser Arg Cys Trp Val Ala Val Thr Pro Thr Val Ala Thr Arg Asp 20 25 30 Gly Lys Leu Pro Thr Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val 35 40 45 Gly Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly 50 55 60 Ser Val Phe Leu Val Gly Gln Leu Phe Thr Phe Ser Pro Arg Arg His 65 70 75 80 Trp Thr Thr Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr 85 90 95 Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Ala 100 105 110 Ala Leu 80 91 PRT Hepatitis C virus 80 Gly Val Asp Ala Glu Thr His Val Thr Gly Gly Asn Ala Gly Arg Thr 1 5 10 15 Thr Ala Gly Leu Val Gly Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile 20 25 30 Gln Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn Ser Thr Ala Leu 35 40 45 Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr 50 55 60 Gln His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys 65 70 75 80 Arg Arg Leu Thr Asp Phe Ala Gln Gly Trp Gly 85 90 81 176 PRT Hepatitis C virus 81 Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly Leu Asp Glu Arg Pro 1 5 10 15 Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val Pro Ala Lys 20 25 30 Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val 35 40 45 Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Ala Asn 50 55 60 Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn 65 70 75 80 Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys 85 90 95 Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly Asn Asn Thr Leu Leu 100 105 110 Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Ser Arg 115 120 125 Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Met Val Asp Tyr Pro 130 135 140 Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr Ile Phe Lys 145 150 155 160 Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys 165 170 175 82 96 PRT Hepatitis C virus 82 Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val Pro Ala Lys Ser Val 1 5 10 15 Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr 20 25 30 Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Ala Asn Asp Thr 35 40 45 Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe 50 55 60 Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys Gly Ala 65 70 75 80 Pro Pro Cys Val Ile Gly Gly Val Gly Asn Asn Thr Leu Leu Cys Pro 85 90 95 83 278 PRT Hepatitis C virus 83 Ala Ala Cys Gly Asp Ile Ile Asn Gly Leu Pro Val Ser Ala Arg Arg 1 5 10 15 Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Val Ser Lys Gly 20 25 30 Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly 35 40 45 Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln 50 55 60 Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Thr Gln Thr Phe Leu 65 70 75 80 Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly 85 90 95 Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr 100 105 110 Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser Arg 115 120 125 Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr 130 135 140 Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly 145 150 155 160 Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly 165 170 175 Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Leu Phe Arg Ala 180 185 190 Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val 195 200 205 Glu Asn Leu Gly Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser 210 215 220 Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala 225 230 235 240 Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala 245 250 255 Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu 260 265 270 Gly Phe Gly Ala Tyr Met 275 84 158 PRT Hepatitis C virus 84 Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile 1 5 10 15 Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly 20 25 30 His Ala Val Gly Leu Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala 35 40 45 Lys Ala Val Asp Phe Ile Pro Val Glu Asn Leu Gly Thr Thr Met Arg 50 55 60 Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser 65 70 75 80 Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr 85 90 95 Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu 100 105 110 Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys 115 120 125 Ala His Gly Val Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr 130 135 140 Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu 145 150 155 85 263 PRT Hepatitis C virus 85 Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys 1 5 10 15 Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly 20 25 30 Leu Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp 35 40 45 Phe Ile Pro Val Glu Asn Leu Gly Thr Thr Met Arg Ser Pro Val Phe 50 55 60 Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala 65 70 75 80 His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala 85 90 95 Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val 100 105 110 Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val 115 120 125 Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro 130 135 140 Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser 145 150 155 160 Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp 165 170 175 Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr 180 185 190 Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser 195 200 205 Val Thr Val Ser His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr 210 215 220 Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys 225 230 235 240 Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu 245 250 255 Leu Ala Ala Lys Leu Val Ala 260 86 194 PRT Hepatitis C virus 86 Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His 1 5 10 15 Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala 20 25 30 Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala 35 40 45 Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp 50 55 60 Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile 65 70 75 80 Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly 85 90 95 Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala 100 105 110 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 115 120 125 Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val 130 135 140 Thr Val Ser His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly 145 150 155 160 Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly 165 170 175 Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu 180 185 190 Ala Ala 87 205 PRT Hepatitis C virus 87 Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys 1 5 10 15 Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr 20 25 30 Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala 35 40 45 Thr Ala Thr Pro Pro Gly Ser Val Thr Val Ser His Pro Asn Ile Glu 50 55 60 Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala 65 70 75 80 Ile Pro Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His 85 90 95 Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly 100 105 110 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro 115 120 125 Thr Ser Gly Asp Val Val Val Val Ser Thr Asp Ala Leu Met Thr Gly 130 135 140 Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr 145 150 155 160 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr 165 170 175 Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr 180 185 190 Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro 195 200 205 88 186 PRT Hepatitis C virus 88 Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln 1 5 10 15 Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro 20 25 30 Pro Gly Ser Val Thr Val Ser His Pro Asn Ile Glu Glu Val Ala Leu 35 40 45 Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu 50 55 60 Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys 65 70 75 80 Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val 85 90 95 Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp 100 105 110 Val Val Val Val Ser Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp 115 120 125 Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp 130 135 140 Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gln 145 150 155 160 Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys 165 170 175 Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly 180 185 89 158 PRT Hepatitis C virus 89 Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu 1 5 10 15 Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gln Asp Ala Val 20 25 30 Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile 35 40 45 Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser 50 55 60 Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu 65 70 75 80 Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro 85 90 95 Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe 100 105 110 Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln 115 120 125 Ser Gly Glu Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys 130 135 140 Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp 145 150 155 90 129 PRT Hepatitis C virus 90 Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser 1 5 10 15 Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr 20 25 30 Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly 35 40 45 Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr 50 55 60 Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser 65 70 75 80 Gly Glu Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala 85 90 95 Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu 100 105 110 Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg 115 120 125 Leu 91 51 PRT Hepatitis C virus 91 Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala 1 5 10 15 Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu 20 25 30 Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu 35 40 45 Phe Asp Glu 50 92 18 PRT Hepatitis C virus 92 Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val 1 5 10 15 Gly Arg 93 208 PRT Hepatitis C virus 93 Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Gly Gln Thr Leu Leu 1 5 10 15 Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala Pro Gly 20 25 30 Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly 35 40 45 Ser Val Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly 50 55 60 Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu 65 70 75 80 Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser 85 90 95 Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg 100 105 110 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile 115 120 125 Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro 130 135 140 Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr 145 150 155 160 Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys 165 170 175 Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile 180 185 190 Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met 195 200 205 94 207 PRT Hepatitis C virus 94 Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly Ser Val 1 5 10 15 Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly 20 25 30 Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro 35 40 45 Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly 50 55 60 Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 65 70 75 80 Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe 85 90 95 Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser 100 105 110 Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr 115 120 125 Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr 130 135 140 Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu 145 150 155 160 Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln 165 170 175 Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg Gly Tyr Arg Gly Val 180 185 190 Trp Arg Gly Asp Gly Ile Met His Thr Arg Cys His Cys Gly Ala 195 200 205 95 225 PRT Hepatitis C virus 95 Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu 1 5 10 15 Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu 20 25 30 Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly 35 40 45 Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly 50 55 60 Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn 65 70 75 80 His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg 85 90 95 Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg 100 105 110 Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser 115 120 125 Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe 130 135 140 Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro 145 150 155 160 Phe Val Ser Cys Gln Arg Gly Tyr Arg Gly Val Trp Arg Gly Asp Gly 165 170 175 Ile Met His Thr Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val 180 185 190 Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met 195 200 205 Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr 210 215 220 Pro 225 96 145 PRT Hepatitis C virus 96 Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Ile 1 5 10 15 Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro 20 25 30 Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala 35 40 45 Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met 50 55 60 Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr 65 70 75 80 His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile Leu 85 90 95 Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile 100 105 110 Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile 115 120 125 Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys 130 135 140 Ala 145 97 54 PRT Hepatitis C virus 97 Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val 1 5 10 15 Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe 20 25 30 Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser 35 40 45 Asp Ala Ala Ala Arg Val 50 98 165 PRT Hepatitis C virus 98 Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser 1 5 10 15 Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr 20 25 30 Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr 35 40 45 Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu 50 55 60 Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln 65 70 75 80 Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg Gly Tyr Arg Gly Val 85 90 95 Trp Arg Gly Asp Gly Ile Met His Thr Arg Cys His Cys Gly Ala Glu 100 105 110 Ile Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg 115 120 125 Thr Cys Arg Asn Met Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr 130 135 140 Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn Tyr Lys Phe Ala Leu 145 150 155 160 Trp Arg Val Ser Ala 165 99 308 PRT Hepatitis C virus 99 Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser 1 5 10 15 Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser 20 25 30 Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp 35 40 45 Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala 50 55 60 Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg 65 70 75 80 Gly Tyr Arg Gly Val Trp Arg Gly Asp Gly Ile Met His Thr Arg Cys 85 90 95 His Cys Gly Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr Met Arg 100 105 110 Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Thr Phe Pro 115 120 125 Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn 130 135 140 Tyr Lys Phe Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Val Glu Ile 145 150 155 160 Arg Arg Val Gly Asp Phe His Tyr Val Ser Gly Met Thr Thr Asp Asn 165 170 175 Leu Lys Cys Pro Cys Gln Ile Pro Ser Pro Glu Phe Phe Thr Glu Leu 180 185 190 Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu 195 200 205 Arg Glu Glu Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro Val Gly 210 215 220 Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser 225 230 235 240 Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg 245 250 255 Leu Ala Arg Gly Ser Pro Pro Ser Met Ala Ser Ser Ser Ala Ser Gln 260 265 270 Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn His Asp Ser 275 280 285 Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met 290 295 300 Gly Gly Asn Ile 305 100 283 PRT Hepatitis C virus 100 Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp 1 5 10 15 Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp 20 25 30 Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu 35 40 45 Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys 50 55 60 Gln Arg Gly Tyr Arg Gly Val Trp Arg Gly Asp Gly Ile Met His Thr 65 70 75 80 Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr 85 90 95 Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Thr 100 105 110 Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala 115 120 125 Pro Asn Tyr Lys Phe Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Val 130 135 140 Glu Ile Arg Arg Val Gly Asp Phe His Tyr Val Ser Gly Met Thr Thr 145 150 155 160 Asp Asn Leu Lys Cys Pro Cys Gln Ile Pro Ser Pro Glu Phe Phe Thr 165 170 175 Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro 180 185 190 Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro 195 200 205 Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu 210 215 220 Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly 225 230 235 240 Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Met Ala Ser Ser Ser Ala 245 250 255 Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn His 260 265 270 Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn 275 280 101 249 PRT Hepatitis C virus 101 Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser 1 5 10 15 Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp 20 25 30 Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala 35 40 45 Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg 50 55 60 Gly Tyr Arg Gly Val Trp Arg Gly Asp Gly Ile Met His Thr Arg Cys 65 70 75 80 His Cys Gly Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr Met Arg 85 90 95 Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Thr Phe Pro 100 105 110 Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn 115 120 125 Tyr Lys Phe Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Val Glu Ile 130 135 140 Arg Arg Val Gly Asp Phe His Tyr Val Ser Gly Met Thr Thr Asp Asn 145 150 155 160 Leu Lys Cys Pro Cys Gln Ile Pro Ser Pro Glu Phe Phe Thr Glu Leu 165 170 175 Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu 180 185 190 Arg Glu Glu Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro Val Gly 195 200 205 Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser 210 215 220 Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg 225 230 235 240 Leu Ala Arg Gly Ser Pro Pro Ser Met 245 102 85 PRT Hepatitis C virus 102 Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe 1 5 10 15 Val Ser Cys Gln Arg Gly Tyr Arg Gly Val Trp Arg Gly Asp Gly Ile 20 25 30 Met His Thr Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys 35 40 45 Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp 50 55 60 Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro 65 70 75 80 Leu Pro Ala Pro Asn 85 103 94 PRT Hepatitis C virus 103 Glu Ile Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro 1 5 10 15 Arg Thr Cys Arg Asn Met Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr 20 25 30 Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn Tyr Lys Phe Ala 35 40 45 Leu Trp Arg Val Ser Ala Glu Glu Tyr Val Glu Ile Arg Arg Val Gly 50 55 60 Asp Phe His Tyr Val Ser Gly Met Thr Thr Asp Asn Leu Lys Cys Pro 65 70 75 80 Cys Gln Ile Pro Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly 85 90 104 75 PRT Hepatitis C virus 104 Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr 1 5 10 15 Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro 20 25 30 Leu Val Ala Glu Glu Asp Glu Arg Glu Val Ser Val Pro Ala Glu Ile 35 40 45 Leu Arg Lys Ser Arg Arg Phe Ala Arg Ala Leu Pro Val Trp Ala Arg 50 55 60 Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp 65 70 75 105 90 PRT Hepatitis C virus 105 His Gly Cys Pro Leu Pro Pro Pro Arg Ser Pro Pro Val Pro Pro Pro 1 5 10 15 Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Ser Thr Ala 20 25 30 Leu Ala Glu Leu Ala Thr Lys Ser Phe Gly Ser Ser Ser Thr Ser Gly 35 40 45 Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly 50 55 60 Cys Pro Pro Asp Ser Asp Val Glu Ser Tyr Ser Ser Met Pro Pro Leu 65 70 75 80 Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser 85 90 106 137 PRT Hepatitis C virus 106 Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys 1 5 10 15 Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu 20 25 30 Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg Gln Lys Lys Val 35 40 45 Thr Phe Asp Arg Leu Gln Val Leu Asp Ser His Tyr Gln Asp Val Leu 50 55 60 Lys Glu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser 65 70 75 80 Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys 85 90 95 Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val 100 105 110 Ala His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Ser Val Thr 115 120 125 Pro Ile Asp Thr Thr Ile Met Ala Lys 130 135 107 300 PRT Hepatitis C virus 107 Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His 1 5 10 15 Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His 20 25 30 Ala Arg Lys Ala Val Ala His Ile Asn Ser Val Trp Lys Asp Leu Leu 35 40 45 Glu Asp Ser Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu 50 55 60 Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu 65 70 75 80 Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu 85 90 95 Tyr Asp Val Val Ser Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr 100 105 110 Gly Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala 115 120 125 Trp Lys Ser Lys Lys Thr Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys 130 135 140 Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile 145 150 155 160 Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala Ile Lys Ser 165 170 175 Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly 180 185 190 Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr 195 200 205 Ser Cys Gly Asn Thr Leu Thr Cys Tyr Ile Lys Ala Arg Ala Ala Cys 210 215 220 Arg Ala Ala Gly Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp 225 230 235 240 Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp Ala Ala Ser 245 250 255 Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly 260 265 270 Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser 275 280 285 Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys 290 295 300 108 199 PRT Hepatitis C virus 108 Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe 1 5 10 15 Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Ala 20 25 30 His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Ser Val Thr Pro 35 40 45 Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro 50 55 60 Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu 65 70 75 80 Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Ser Lys 85 90 95 Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro 100 105 110 Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys Thr 115 120 125 Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr 130 135 140 Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp Leu 145 150 155 160 Asp Pro Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr 165 170 175 Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg 180 185 190 Arg Cys Arg Ala Ser Gly Val 195 109 260 PRT Hepatitis C virus 109 Leu Leu Glu Asp Ser Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys 1 5 10 15 Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala 20 25 30 Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met 35 40 45 Ala Leu Tyr Asp Val Val Ser Lys Leu Pro Leu Ala Val Met Gly Ser 50 55 60 Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val 65 70 75 80 Gln Ala Trp Lys Ser Lys Lys Thr Pro Met Gly Phe Ser Tyr Asp Thr 85 90 95 Arg Cys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Thr Glu Glu 100 105 110 Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala Ile 115 120 125 Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn Ser 130 135 140 Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu 145 150 155 160 Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Ile Lys Ala Arg Ala 165 170 175 Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys Thr Met Leu Val Cys Gly 180 185 190 Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp Ala 195 200 205 Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro 210 215 220 Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser 225 230 235 240 Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Arg Val 245 250 255 Tyr Tyr Leu Thr 260 110 127 PRT Hepatitis C virus 110 Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp Ala Ala Ser Leu Arg 1 5 10 15 Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro 20 25 30 Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn 35 40 45 Val Ser Val Ala His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr 50 55 60 Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg 65 70 75 80 His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro 85 90 95 Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Val Leu 100 105 110 Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asn Cys Glu Ile Tyr 115 120 125 111 89 PRT Hepatitis C virus 111 Val Ser Val Ala His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr 1 5 10 15 Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg 20 25 30 His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro 35 40 45 Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Val Leu 50 55 60 Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asn Cys Glu Ile Tyr Gly 65 70 75 80 Ala Cys Tyr Ser Ile Glu Pro Leu Asp 85 112 73 PRT Hepatitis C virus 112 Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu 1 5 10 15 Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu 20 25 30 Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ser 35 40 45 Arg Gly Gly Arg Ala Ala Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala 50 55 60 Val Arg Thr Lys Leu Lys Leu Thr Pro 65 70 113 63 PRT Hepatitis C virus 113 Ser Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu Gly 1 5 10 15 Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg Ala 20 25 30 Arg Leu Leu Ser Arg Gly Gly Arg Ala Ala Ile Cys Gly Lys Tyr Leu 35 40 45 Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Ala 50 55 60 114 310 DNA Hepatitis C virus 114 tgcttgcgag tgccccggga ggtctcgtag accgtgcacc atgagcacga atcctaaacc 60 tcaaagaaaa accaaacgta acaccaaccg tcgcccacag gacgtcaagt tcccgggtgg 120 cggtcagatc gttggtggag tttacttgtt gccgcgcagg ggccctagat tgggtgtgcg 180 cgcgacgagg aagacttccg agcggtcgca acctcgaggt agacgtcagc ctatccccaa 240 ggcacgtcgg cccgagggca ggacctgggc tcagcccggg tacccttggc ccctctatgg 300 caatgagggt 310 115 339 DNA Hepatitis C virus 115 atgagcacga atcctaaacc tcaaagaaaa accaaacgta acaccaaccg tcgcccacag 60 gacgtcaagt tcccgggtgg cggtcagatc gttggtggag tttacttgtt gccgcgcagg 120 ggccctagat tgggtgtgcg cgcgacgagg aagacttccg agcggtcgca acctcgaggt 180 agacgtcagc ctatccccaa ggcacgtcgg cccgagggca ggacctgggc tcagcccggg 240 tacccttggc ccctctatgg caatgagggt tgcgggtggg cgggatggct cctgtctccc 300 cgtggctctc ggcctagctg gggccccaca gacccccgg 339 116 345 DNA Hepatitis C virus 116 tgccatcctg cacactccgg ggtgtgtccc ttgcgttcgc gagggtaacg cctcgaggtg 60 ttgggtggcg gtgaccccca cggtggccac cagggacggc aaactcccca caacgcagct 120 tcgacgtcat atcgatctgc ttgtcgggag cgccaccctc tgctcggccc tctacgtggg 180 ggacctgtgc gggtctgtct ttcttgttgg tcaactgttt accttctctc ccaggcgcca 240 ctggacgacg caagactgca attgttctat ctatcccggc catataacgg gtcatcgcat 300 ggcatgggat atgatgatga actggtcccc tacggcagcg ttggt 345 117 276 DNA Hepatitis C virus 117 cggcgtcgac gcggaaaccc acgtcaccgg gggaaatgcc ggccgcacca cggctgggct 60 tgttggtctc cttacaccag gcgccaagca gaacatccaa ctgatcaaca ccaacggcag 120 ttggcacatc aatagcacgg ccttgaattg caatgaaagc cttaacaccg gctggttagc 180 agggctcttc tatcaacaca aattcaactc ttcaggctgt cctgagaggt tggccagctg 240 ccgacgcctt accgattttg cccagggctg gggtcc 276 118 531 DNA Hepatitis C virus 118 ctggggtcct atcagttatg ccaacggaag cggcctcgac gaacgcccct actgctggca 60 ctaccctcca agaccttgtg gcattgtgcc cgcaaagagc gtgtgtggcc cggtatattg 120 cttcactccc agccccgtgg tggtgggaac gaccgacagg tcgggcgcgc ctacctacag 180 ctggggtgca aatgatacgg atgtcttcgt ccttaacaac accaggccac cgctgggcaa 240 ttggttcggt tgtacctgga tgaactcaac tggattcacc aaagtgtgcg gagcgccccc 300 ttgtgtcatc ggaggggtgg gcaacaacac cttgctctgc cccactgatt gcttccgcaa 360 acatccggaa gccacatact ctcggtgcgg ctccggtccc tggattacac ccaggtgcat 420 ggtcgactac ccgtataggc tttggcacta tccttgtacc atcaattaca ccatattcaa 480 agtcaggatg tacgtgggag gggtcgagca caggctggaa gcggcctgca a 531 119 289 DNA Hepatitis C virus 119 ctggcactac cctccaagac cttgtggcat tgtgcccgca aagagcgtgt gtggcccggt 60 atattgcttc actcccagcc ccgtggtggt gggaacgacc gacaggtcgg gcgcgcctac 120 ctacagctgg ggtgcaaatg atacggatgt cttcgtcctt aacaacacca ggccaccgct 180 gggcaattgg ttcggttgta cctggatgaa ctcaactgga ttcaccaaag tgtgcggagc 240 gcccccttgt gtcatcggag gggtgggcaa caacaccttg ctctgcccc 289 120 836 DNA Hepatitis C virus 120 gccgcgtgcg gtgacatcat caacggcttg cccgtctctg cccgtagggg ccaggagata 60 ctgcttgggc cagccgacgg aatggtctcc aaggggtgga ggttgctggc gcccatcacg 120 gcgtacgccc agcagacgag aggcctccta gggtgtataa tcaccagcct gactggccgg 180 gacaaaaacc aagtggaggg tgaggtccag atcgtgtcaa ctgctaccca aaccttcctg 240 gcaacgtgca tcaatggggt atgctggact gtctaccacg gggccggaac gaggaccatc 300 gcatcaccca agggtcctgt catccagatg tataccaatg tggaccaaga ccttgtgggc 360 tggcccgctc ctcaaggttc ccgctcattg acaccctgta cctgcggctc ctcggacctt 420 tacctggtca cgaggcacgc cgatgtcatt cccgtgcgcc ggcgaggtga tagcaggggt 480 agcctgcttt cgccccggcc catttcctac ttgaaaggct cctcgggggg tccgctgttg 540 tgccccgcgg gacacgccgt gggcctattc agggccgcgg tgtgcacccg tggagtggct 600 aaagcggtgg actttatccc tgtggagaac ctagggacaa ccatgagatc cccggtgttc 660 acggacaact cctctccacc agcagtgccc cagagcttcc aggtggccca cctgcatgct 720 cccaccggca gcggtaagag caccaaggtc ccggctgcgt acgcagccca gggctacaag 780 gtgttggtgc tcaacccctc tgttgctgca acgctgggct ttggtgctta catgtc 836 121 475 DNA Hepatitis C virus 121 gcgccggcga ggtgatagca ggggtagcct gctttcgccc cggcccattt cctacttgaa 60 aggctcctcg gggggtccgc tgttgtgccc cgcgggacac gccgtgggcc tattcagggc 120 cgcggtgtgc acccgtggag tggctaaagc ggtggacttt atccctgtgg agaacctagg 180 gacaaccatg agatccccgg tgttcacgga caactcctct ccaccagcag tgccccagag 240 cttccaggtg gcccacctgc atgctcccac cggcagcggt aagagcacca aggtcccggc 300 tgcgtacgca gcccagggct acaaggtgtt ggtgctcaac ccctctgttg ctgcaacgct 360 gggctttggt gcttacatgt ccaaggccca tggggttgat cctaatatca ggaccggggt 420 gagaacaatt accactggca gccccatcac gtactccacc tacggcaagt tcctt 475 122 790 DNA Hepatitis C virus 122 tgatagcagg ggtagcctgc tttcgccccg gcccatttcc tacttgaaag gctcctcggg 60 gggtccgctg ttgtgccccg cgggacacgc cgtgggccta ttcagggccg cggtgtgcac 120 ccgtggagtg gctaaagcgg tggactttat ccctgtggag aacctaggga caaccatgag 180 atccccggtg ttcacggaca actcctctcc accagcagtg ccccagagct tccaggtggc 240 ccacctgcat gctcccaccg gcagcggtaa gagcaccaag gtcccggctg cgtacgcagc 300 ccagggctac aaggtgttgg tgctcaaccc ctctgttgct gcaacgctgg gctttggtgc 360 ttacatgtcc aaggcccatg gggttgatcc taatatcagg accggggtga gaacaattac 420 cactggcagc cccatcacgt actccaccta cggcaagttc cttgccgacg gcgggtgctc 480 aggaggtgct tatgacataa taatttgtga cgagtgccac tccacggatg ccacatccat 540 cttgggcatc ggcactgtcc ttgaccaagc agagactgcg ggggcgagac tggttgtgct 600 cgccactgct acccctccgg gctccgtcac tgtgtcccat cctaacatcg aggaggttgc 660 tctgtccacc accggagaga tcccctttta cggcaaggct atccccctcg aggtgatcaa 720 ggggggaaga catctcatct tctgccactc aaagaagaag tgcgacgagc tcgccgcgaa 780 gctggtcgca 790 123 583 DNA Hepatitis C virus 123 ggacaactcc tctccaccag cagtgcccca gagcttccag gtggcccacc tgcatgctcc 60 caccggcagc ggtaagagca ccaaggtccc ggctgcgtac gcagcccagg gctacaaggt 120 gttggtgctc aacccctctg ttgctgcaac gctgggcttt ggtgcttaca tgtccaaggc 180 ccatggggtt gatcctaata tcaggaccgg ggtgagaaca attaccactg gcagccccat 240 cacgtactcc acctacggca agttccttgc cgacggcggg tgctcaggag gtgcttatga 300 cataataatt tgtgacgagt gccactccac ggatgccaca tccatcttgg gcatcggcac 360 tgtccttgac caagcagaga ctgcgggggc gagactggtt gtgctcgcca ctgctacccc 420 tccgggctcc gtcactgtgt cccatcctaa catcgaggag gttgctctgt ccaccaccgg 480 agagatcccc ttttacggca aggctatccc cctcgaggtg atcaaggggg gaagacatct 540 catcttctgc cactcaaaga agaagtgcga cgagctcgcc gcg 583 124 617 DNA Hepatitis C virus 124 ccttgccgac ggcgggtgct caggaggtgc ttatgacata ataatttgtg acgagtgcca 60 ctccacggat gccacatcca tcttgggcat cggcactgtc cttgaccaag cagagactgc 120 gggggcgaga ctggttgtgc tcgccactgc tacccctccg ggctccgtca ctgtgtccca 180 tcctaacatc gaggaggttg ctctgtccac caccggagag atcccctttt acggcaaggc 240 tatccccctc gaggtgatca aggggggaag acatctcatc ttctgccact caaagaagaa 300 gtgcgacgag ctcgccgcga agctggtcgc attgggcatc aatgccgtgg cctactaccg 360 cggtcttgac gtgtctgtca tcccgaccag cggcgatgtt gtcgtcgtgt cgaccgatgc 420 tctcatgact ggctttaccg gcgacttcga ctctgtgata gactgcaaca cgtgtgtcac 480 tcagacagtc gatttcagcc ttgaccctac ctttaccatt gagacaacca cgctccccca 540 ggatgctgtc tccaggactc aacgccgggg caggactggc agggggaagc caggcatcta 600 tagatttgtg gcaccgg 617 125 559 DNA Hepatitis C virus 125 ctccacggat gccacatcca tcttgggcat cggcactgtc cttgaccaag cagagactgc 60 gggggcgaga ctggttgtgc tcgccactgc tacccctccg ggctccgtca ctgtgtccca 120 tcctaacatc gaggaggttg ctctgtccac caccggagag atcccctttt acggcaaggc 180 tatccccctc gaggtgatca aggggggaag acatctcatc ttctgccact caaagaagaa 240 gtgcgacgag ctcgccgcga agctggtcgc attgggcatc aatgccgtgg cctactaccg 300 cggtcttgac gtgtctgtca tcccgaccag cggcgatgtt gtcgtcgtgt cgaccgatgc 360 tctcatgact ggctttaccg gcgacttcga ctctgtgata gactgcaaca cgtgtgtcac 420 tcagacagtc gatttcagcc ttgaccctac ctttaccatt gagacaacca cgctccccca 480 ggatgctgtc tccaggactc aacgccgggg caggactggc agggggaagc caggcatcta 540 tagatttgtg gcaccgggg 559 126 475 DNA Hepatitis C virus 126 tgtgatagac tgcaacacgt gtgtcactca gacagtcgat ttcagccttg accctacctt 60 taccattgag acaaccacgc tcccccagga tgctgtctcc aggactcaac gccggggcag 120 gactggcagg gggaagccag gcatctatag atttgtggca ccgggggagc gcccctccgg 180 catgttcgac tcgtccgtcc tctgtgagtg ctatgacgcg ggctgtgctt ggtatgagct 240 cacgcccgcc gagactacag ttaggctacg agcgtacatg aacaccccgg ggcttcccgt 300 gtgccaggac catcttgaat tttgggaggg cgtctttacg ggcctcactc atatagatgc 360 ccacttttta tcccagacaa agcagagtgg ggagaacttt ccttacctgg tagcgtacca 420 agccaccgtg tgcgctaggg ctcaagcccc tcccccatcg tgggaccaga tgtgg 475 127 390 DNA Hepatitis C virus 127 tagatttgtg gcaccggggg agcgcccctc cggcatgttc gactcgtccg tcctctgtga 60 gtgctatgac gcgggctgtg cttggtatga gctcacgccc gccgagacta cagttaggct 120 acgagcgtac atgaacaccc cggggcttcc cgtgtgccag gaccatcttg aattttggga 180 gggcgtcttt acgggcctca ctcatataga tgcccacttt ttatcccaga caaagcagag 240 tggggagaac tttccttacc tggtagcgta ccaagccacc gtgtgcgcta gggctcaagc 300 ccctccccca tcgtgggacc agatgtggaa gtgtttgatc cgccttaaac ccaccctcca 360 tgggccaaca cccctgctat acagactggg 390 128 155 DNA Hepatitis C virus 128 acgagcacct gggtgctcgt tggcggcgtc ctggctgctc tggccgcgta ttgcctgtca 60 acaggctgcg tggtcatagt gggcaggatc gtcttgtccg ggaagccggc aattatacct 120 gacagggagg ttctctacca ggagttcgat gagat 155 129 56 DNA Hepatitis C virus 129 ggctgctctg gccgcgtatt gcctgtcaac aggctgcgtg gtcatagtgg gcagga 56 130 625 DNA Hepatitis C virus 130 ttttacagct gccgtcacca gcccactaac cactggccaa accctcctct tcaacatatt 60 gggggggtgg gtggctgccc agctcgccgc ccccggtgcc gctactgcct ttgtgggtgc 120 tggcctagct ggcgccgcca tcggcagcgt tggactgggg aaggtcctcg tggacattct 180 tgcagggtat ggcgcgggcg tggcgggagc tcttgtagca ttcaagatca tgagcggtga 240 ggtcccctcc acggaggacc tggtcaatct gctgcccgcc atcctctcgc ctggagccct 300 tgtagtcggt gtggtctgcg cagcaatact gcgccggcac gttggcccgg gcgagggggc 360 agtgcaatgg atgaaccggc taatagcctt cgcctcccgg gggaaccatg tttcccccac 420 gcactacgtg ccggagagcg atgcagccgc ccgcgtcact gccatactca gcagcctcac 480 tgtaacccag ctcctgaggc gactgcatca gtggataagc tcggagtgta ccactccatg 540 ctccggttcc tggctaaggg acatctggga ctggatatgc gaggtgctga gcgactttaa 600 gacctggctg aaagccaagc tcatg 625 131 623 DNA Hepatitis C virus 131 tactgccttt gtgggtgctg gcctagctgg cgccgccatc ggcagcgttg gactggggaa 60 ggtcctcgtg gacattcttg cagggtatgg cgcgggcgtg gcgggagctc ttgtagcatt 120 caagatcatg agcggtgagg tcccctccac ggaggacctg gtcaatctgc tgcccgccat 180 cctctcgcct ggagcccttg tagtcggtgt ggtctgcgca gcaatactgc gccggcacgt 240 tggcccgggc gagggggcag tgcaatggat gaaccggcta atagccttcg cctcccgggg 300 gaaccatgtt tcccccacgc actacgtgcc ggagagcgat gcagccgccc gcgtcactgc 360 catactcagc agcctcactg taacccagct cctgaggcga ctgcatcagt ggataagctc 420 ggagtgtacc actccatgct ccggttcctg gctaagggac atctgggact ggatatgcga 480 ggtgctgagc gactttaaga cctggctgaa agccaagctc atgccacaac tgcctgggat 540 tccctttgtg tcctgccagc gcgggtatag gggggtctgg cgaggagacg gcattatgca 600 cactcgctgc cactgtggag ctg 623 132 678 DNA Hepatitis C virus 132 cctcgtggac attcttgcag ggtatggcgc gggcgtggcg ggagctcttg tagcattcaa 60 gatcatgagc ggtgaggtcc cctccacgga ggacctggtc aatctgctgc ccgccatcct 120 ctcgcctgga gcccttgtag tcggtgtggt ctgcgcagca atactgcgcc ggcacgttgg 180 cccgggcgag ggggcagtgc aatggatgaa ccggctaata gccttcgcct cccgggggaa 240 ccatgtttcc cccacgcact acgtgccgga gagcgatgca gccgcccgcg tcactgccat 300 actcagcagc ctcactgtaa cccagctcct gaggcgactg catcagtgga taagctcgga 360 gtgtaccact ccatgctccg gttcctggct aagggacatc tgggactgga tatgcgaggt 420 gctgagcgac tttaagacct ggctgaaagc caagctcatg ccacaactgc ctgggattcc 480 ctttgtgtcc tgccagcgcg ggtatagggg ggtctggcga ggagacggca ttatgcacac 540 tcgctgccac tgtggagctg agatcactgg acatgtcaaa aacgggacga tgaggatcgt 600 cggtcctagg acctgcagga acatgtggag tgggacgttc cccattaacg cctacaccac 660 gggcccctgt actcccct 678 133 436 DNA Hepatitis C virus 133 tgcagggtat ggcgcgggcg tggcgggagc tcttgtagca ttcaagatca tgagcggtga 60 ggtcccctcc acggaggacc tggtcaatct gctgcccgcc atcctctcgc ctggagccct 120 tgtagtcggt gtggtctgcg cagcaatact gcgccggcac gttggcccgg gcgagggggc 180 agtgcaatgg atgaaccggc taatagcctt cgcctcccgg gggaaccatg tttcccccac 240 gcactacgtg ccggagagcg atgcagccgc ccgcgtcact gccatactca gcagcctcac 300 tgtaacccag ctcctgaggc gactgcatca gtggataagc tcggagtgta ccactccatg 360 ctccggttcc tggctaaggg acatctggga ctggatatgc gaggtgctga gcgactttaa 420 gacctggctg aaagcc 436 134 164 DNA Hepatitis C virus 134 agcccttgta gtcggtgtgg tctgcgcagc aatactgcgc cggcacgttg gcccgggcga 60 gggggcagtg caatggatga accggctaat agccttcgcc tcccggggga accatgtttc 120 ccccacgcac tacgtgccgg agagcgatgc agccgcccgc gtca 164 135 496 DNA Hepatitis C virus 135 cgcctcccgg gggaaccatg tttcccccac gcactacgtg ccggagagcg atgcagccgc 60 ccgcgtcact gccatactca gcagcctcac tgtaacccag ctcctgaggc gactgcatca 120 gtggataagc tcggagtgta ccactccatg ctccggttcc tggctaaggg acatctggga 180 ctggatatgc gaggtgctga gcgactttaa gacctggctg aaagccaagc tcatgccaca 240 actgcctggg attccctttg tgtcctgcca gcgcgggtat aggggggtct ggcgaggaga 300 cggcattatg cacactcgct gccactgtgg agctgagatc actggacatg tcaaaaacgg 360 gacgatgagg atcgtcggtc ctaggacctg caggaacatg tggagtggga cgttccccat 420 taacgcctac accacgggcc cctgtactcc ccttcctgcg ccgaactata agttcgcgct 480 gtggagggtg tctgca 496 136 926 DNA Hepatitis C virus 136 tacgtgccgg agagcgatgc agccgcccgc gtcactgcca tactcagcag cctcactgta 60 acccagctcc tgaggcgact gcatcagtgg ataagctcgg agtgtaccac tccatgctcc 120 ggttcctggc taagggacat ctgggactgg atatgcgagg tgctgagcga ctttaagacc 180 tggctgaaag ccaagctcat gccacaactg cctgggattc cctttgtgtc ctgccagcgc 240 gggtataggg gggtctggcg aggagacggc attatgcaca ctcgctgcca ctgtggagct 300 gagatcactg gacatgtcaa aaacgggacg atgaggatcg tcggtcctag gacctgcagg 360 aacatgtgga gtgggacgtt ccccattaac gcctacacca cgggcccctg tactcccctt 420 cctgcgccga actataagtt cgcgctgtgg agggtgtctg cagaggaata cgtggagata 480 aggcgggtgg gggacttcca ctacgtatcg ggtatgacta ctgacaatct taaatgcccg 540 tgccagatcc catcgcccga atttttcaca gaattggacg gggtgcgcct acacaggttt 600 gcgccccctt gcaagccctt gctgcgggag gaggtatcat tcagagtagg actccacgag 660 tacccggtgg ggtcgcaatt accttgcgag cccgaaccgg acgtagccgt gttgacgtcc 720 atgctcactg atccctccca tataacagca gaggcggccg ggagaaggtt ggcgagaggg 780 tcaccccctt ctatggccag ctcctcggct agccagctgt ccgctccatc tctcaaggca 840 acttgcaccg ccaaccatga ctcccctgac gccgagctca tagaggctaa cctcctgtgg 900 aggcaggaga tgggcggcaa catcac 926 137 850 DNA Hepatitis C virus 137 actcagcagc ctcactgtaa cccagctcct gaggcgactg catcagtgga taagctcgga 60 gtgtaccact ccatgctccg gttcctggct aagggacatc tgggactgga tatgcgaggt 120 gctgagcgac tttaagacct ggctgaaagc caagctcatg ccacaactgc ctgggattcc 180 ctttgtgtcc tgccagcgcg ggtatagggg ggtctggcga ggagacggca ttatgcacac 240 tcgctgccac tgtggagctg agatcactgg acatgtcaaa aacgggacga tgaggatcgt 300 cggtcctagg acctgcagga acatgtggag tgggacgttc cccattaacg cctacaccac 360 gggcccctgt actccccttc ctgcgccgaa ctataagttc gcgctgtgga gggtgtctgc 420 agaggaatac gtggagataa ggcgggtggg ggacttccac tacgtatcgg gtatgactac 480 tgacaatctt aaatgcccgt gccagatccc atcgcccgaa tttttcacag aattggacgg 540 ggtgcgccta cacaggtttg cgcccccttg caagcccttg ctgcgggagg aggtatcatt 600 cagagtagga ctccacgagt acccggtggg gtcgcaatta ccttgcgagc ccgaaccgga 660 cgtagccgtg ttgacgtcca tgctcactga tccctcccat ataacagcag aggcggccgg 720 gagaaggttg gcgagagggt cacccccttc tatggccagc tcctcggcta gccagctgtc 780 cgctccatct ctcaaggcaa cttgcaccgc caaccatgac tcccctgacg ccgagctcat 840 agaggctaac 850 138 749 DNA Hepatitis C virus 138 cagcctcact gtaacccagc tcctgaggcg actgcatcag tggataagct cggagtgtac 60 cactccatgc tccggttcct ggctaaggga catctgggac tggatatgcg aggtgctgag 120 cgactttaag acctggctga aagccaagct catgccacaa ctgcctggga ttccctttgt 180 gtcctgccag cgcgggtata ggggggtctg gcgaggagac ggcattatgc acactcgctg 240 ccactgtgga gctgagatca ctggacatgt caaaaacggg acgatgagga tcgtcggtcc 300 taggacctgc aggaacatgt ggagtgggac gttccccatt aacgcctaca ccacgggccc 360 ctgtactccc cttcctgcgc cgaactataa gttcgcgctg tggagggtgt ctgcagagga 420 atacgtggag ataaggcggg tgggggactt ccactacgta tcgggtatga ctactgacaa 480 tcttaaatgc ccgtgccaga tcccatcgcc cgaatttttc acagaattgg acggggtgcg 540 cctacacagg tttgcgcccc cttgcaagcc cttgctgcgg gaggaggtat cattcagagt 600 aggactccac gagtacccgg tggggtcgca attaccttgc gagcccgaac cggacgtagc 660 cgtgttgacg tccatgctca ctgatccctc ccatataaca gcagaggcgg ccgggagaag 720 gttggcgaga gggtcacccc cttctatgg 749 139 257 DNA Hepatitis C virus 139 gacctggctg aaagccaagc tcatgccaca actgcctggg attccctttg tgtcctgcca 60 gcgcgggtat aggggggtct ggcgaggaga cggcattatg cacactcgct gccactgtgg 120 agctgagatc actggacatg tcaaaaacgg gacgatgagg atcgtcggtc ctaggacctg 180 caggaacatg tggagtggga cgttccccat taacgcctac accacgggcc cctgtactcc 240 ccttcctgcg ccgaact 257 140 285 DNA Hepatitis C virus 140 tgagatcact ggacatgtca aaaacgggac gatgaggatc gtcggtccta ggacctgcag 60 gaacatgtgg agtgggacgt tccccattaa cgcctacacc acgggcccct gtactcccct 120 tcctgcgccg aactataagt tcgcgctgtg gagggtgtct gcagaggaat acgtggagat 180 aaggcgggtg ggggacttcc actacgtatc gggtatgact actgacaatc ttaaatgccc 240 gtgccagatc ccatcgcccg aatttttcac agaattggac ggggt 285 141 228 DNA Hepatitis C virus 141 catagaggct aacctcctgt ggaggcagga gatgggcggc aacatcacca gggttgagtc 60 agagaacaaa gtggtgattc tggactcctt cgatccgctt gtggcagagg aggatgagcg 120 ggaggtctcc gtacctgcag aaattctgcg gaagtctcgg agattcgccc gggccctgcc 180 cgtctgggcg cggccggact acaacccccc gctagtagag acgtggaa 228 142 273 DNA Hepatitis C virus 142 ccatggctgc ccgctaccac ctccacggtc ccctcctgtg cctccgcctc ggaaaaagcg 60 tacggtggtc ctcaccgaat caaccctatc tactgccttg gccgagcttg ccaccaaaag 120 ttttggcagc tcctcaactt ccggcattac gggcgacaat acgacaacat cctctgagcc 180 cgccccttct ggctgccccc ccgactccga cgttgagtcc tattcttcca tgccccccct 240 ggagggggag cctggggatc cggatctcag cga 273 143 412 DNA Hepatitis C virus 143 ttcctggaca ggcgcactcg tcaccccgtg cgctgcggaa gaacaaaaac tgcccatcaa 60 cgcactgagc aactcgttgc tacgccatca caatctggtg tattccacca cttcacgcag 120 tgcttgccaa aggcagaaga aagtcacatt tgacagactg caagttctgg acagccatta 180 ccaggacgtg ctcaaggagg tcaaagcagc ggcgtcaaaa gtgaaggcta acttgctatc 240 cgtagaggaa gcttgcagcc tgacgccccc acattcagcc aaatccaagt ttggctatgg 300 ggcaaaagac gtccgttgcc atgccagaaa ggccgtagcc cacatcaact ccgtgtggaa 360 agaccttctg gaagacagtg taacaccaat agacactacc atcatggcca ag 412 144 903 DNA Hepatitis C virus 144 ggctaacttg ctatccgtag aggaagcttg cagcctgacg cccccacatt cagccaaatc 60 caagtttggc tatggggcaa aagacgtccg ttgccatgcc agaaaggccg tagcccacat 120 caactccgtg tggaaagacc ttctggaaga cagtgtaaca ccaatagaca ctaccatcat 180 ggccaagaac gaggttttct gcgttcagcc tgagaagggg ggtcgtaagc cagctcgtct 240 catcgtgttc cccgacctgg gcgtgcgcgt gtgcgagaag atggccctgt acgacgtggt 300 tagcaagctc cccctggccg tgatgggaag ctcctacgga ttccaatact caccaggaca 360 gcgggttgaa ttcctcgtgc aagcgtggaa gtccaagaag accccgatgg ggttctcgta 420 tgatacccgc tgttttgact ccacagtcac tgagagcgac atccgtacgg aggaggcaat 480 ttaccaatgt tgtgacctgg acccccaagc ccgcgtggcc atcaagtccc tcactgagag 540 gctttatgtt gggggccctc ttaccaattc aaggggggaa aactgcggct accgcaggtg 600 ccgcgcgagc ggcgtactga caactagctg tggtaacacc ctcacttgct acatcaaggc 660 ccgggcagcc tgtcgagccg cagggctcca ggactgcacc atgctcgtgt gtggcgacga 720 cttagtcgtt atctgtgaaa gtgcgggggt ccaggaggac gcggcgagcc tgagagcctt 780 cacggaggct atgaccaggt actccgcccc ccccggggac cccccacaac cagaatacga 840 cttggagctt ataacatcat gctcctccaa cgtgtcagtc gcccacgacg gcgctggaaa 900 gag 903 145 600 DNA Hepatitis C virus 145 agaggaagct tgcagcctga cgcccccaca ttcagccaaa tccaagtttg gctatggggc 60 aaaagacgtc cgttgccatg ccagaaaggc cgtagcccac atcaactccg tgtggaaaga 120 ccttctggaa gacagtgtaa caccaataga cactaccatc atggccaaga acgaggtttt 180 ctgcgttcag cctgagaagg ggggtcgtaa gccagctcgt ctcatcgtgt tccccgacct 240 gggcgtgcgc gtgtgcgaga agatggccct gtacgacgtg gttagcaagc tccccctggc 300 cgtgatggga agctcctacg gattccaata ctcaccagga cagcgggttg aattcctcgt 360 gcaagcgtgg aagtccaaga agaccccgat ggggttctcg tatgataccc gctgttttga 420 ctccacagtc actgagagcg acatccgtac ggaggaggca atttaccaat gttgtgacct 480 ggacccccaa gcccgcgtgg ccatcaagtc cctcactgag aggctttatg ttgggggccc 540 tcttaccaat tcaagggggg aaaactgcgg ctaccgcagg tgccgcgcga gcggcgtact 600 146 781 DNA Hepatitis C virus 146 ccttctggaa gacagtgtaa caccaataga cactaccatc atggccaaga acgaggtttt 60 ctgcgttcag cctgagaagg ggggtcgtaa gccagctcgt ctcatcgtgt tccccgacct 120 gggcgtgcgc gtgtgcgaga agatggccct gtacgacgtg gttagcaagc tccccctggc 180 cgtgatggga agctcctacg gattccaata ctcaccagga cagcgggttg aattcctcgt 240 gcaagcgtgg aagtccaaga agaccccgat ggggttctcg tatgataccc gctgttttga 300 ctccacagtc actgagagcg acatccgtac ggaggaggca atttaccaat gttgtgacct 360 ggacccccaa gcccgcgtgg ccatcaagtc cctcactgag aggctttatg ttgggggccc 420 tcttaccaat tcaagggggg aaaactgcgg ctaccgcagg tgccgcgcga gcggcgtact 480 gacaactagc tgtggtaaca ccctcacttg ctacatcaag gcccgggcag cctgtcgagc 540 cgcagggctc caggactgca ccatgctcgt gtgtggcgac gacttagtcg ttatctgtga 600 aagtgcgggg gtccaggagg acgcggcgag cctgagagcc ttcacggagg ctatgaccag 660 gtactccgcc ccccccgggg accccccaca accagaatac gacttggagc ttataacatc 720 atgctcctcc aacgtgtcag tcgcccacga cggcgctgga aagagggtct actaccttac 780 c 781 147 382 DNA Hepatitis C virus 147 cgttatctgt gaaagtgcgg gggtccagga ggacgcggcg agcctgagag ccttcacgga 60 ggctatgacc aggtactccg ccccccccgg ggacccccca caaccagaat acgacttgga 120 gcttataaca tcatgctcct ccaacgtgtc agtcgcccac gacggcgctg gaaagagggt 180 ctactacctt acccgtgacc ctacaacccc cctcgcgaga gccgcgtggg agacagcaag 240 acacactcca gtcaattcct ggctaggcaa cataatcatg tttgccccca cactgtgggc 300 gaggatgata ctgatgaccc atttctttag cgtcctcata gccagggatc agcttgaaca 360 ggctcttaac tgtgagatct ac 382 148 268 DNA Hepatitis C virus 148 cgtgtcagtc gcccacgacg gcgctggaaa gagggtctac taccttaccc gtgaccctac 60 aacccccctc gcgagagccg cgtgggagac agcaagacac actccagtca attcctggct 120 aggcaacata atcatgtttg cccccacact gtgggcgagg atgatactga tgacccattt 180 ctttagcgtc ctcatagcca gggatcagct tgaacaggct cttaactgtg agatctacgg 240 agcctgctac tccatagaac cactggat 268 149 222 DNA Hepatitis C virus 149 actccatggc ctcagcgcat tttcactcca cagttactct ccaggtgaaa tcaatagggt 60 ggccgcatgc ctcagaaaac ttggggtccc gcccttgcga gcttggagac accgggcccg 120 gagcgtccgc gctaggcttc tgtccagagg aggcagggct gccatatgtg gcaagtacct 180 cttcaactgg gcagtaagaa caaagctcaa actcactcca at 222 150 192 DNA Hepatitis C virus 150 ctctccaggt gaaatcaata gggtggccgc atgcctcaga aaacttgggg tcccgccctt 60 gcgagcttgg agacaccggg cccggagcgt ccgcgctagg cttctgtcca gaggaggcag 120 ggctgccata tgtggcaagt acctcttcaa ctgggcagta agaacaaagc tcaaactcac 180 tccaatagcg gc 192 151 10 DNA Artificial Sequence Description of Artificial Sequence linker sequence 151 gggccacgaa 10 152 13 DNA Artificial Sequence Description of Artificial Sequence linker sequence 152 ttcgtggccc ctg 13 153 138 DNA Artificial Sequence Description of Artificial Sequence pP6 vector sequence 153 ctagccatgg ccgcaggggc cgcggccgca ctagtgggga tccttaatta aagggccact 60 ggggcccccc gtaccggcgt ccccggcgcc ggcgtgatca cccctaggaa ttaatttccc 120 ggtgaccccg ggggagct 138 154 128 DNA Artificial Sequence Description of Artificial Sequence pB5 vector sequence 154 catggccgca ggggccgcgg ccgcactagt ggggatcctt aattaaaggg ccactggggc 60 cccccggcgt ccccggcgcc ggcgtgatca cccctaggaa ttaatttccc ggtgaccccg 120 ggggagct 128 155 20 DNA Artificial Sequence Description of Artificial Sequence primer 155 gcgtttggaa tcactacagg 20 156 19 DNA Artificial Sequence Description of Artificial Sequence primer 156 cacgatgcac gttgaagtg 19 

1. A nucleic acid which encodes a polypeptide consisting essentially of the amino acid sequences of SEQ ID Nos. 1 to 38 or a sequence complementary thereto.
 2. The nucleic acid sequence according to claim 1, which encodes a polypeptide having at least 95% amino acid identity with the amino acid sequences of SEQ ID Nos. 1 to 38 or a sequence complementary thereto and retains the same binding affinity to said polypeptide of SEQ ID Nos. 1 to
 38. 3. The nucleic acid according to claim 1, wherein said nucleic acid consists essentially of SEQ ID Nos 39 to 76 or a sequence complementary thereto.
 4. The nucleic acid according to claim 3, wherein said nucleic acid has at least 95% nucleic acid identity with a nucleic acid of SEQ ID Nos. 39 to 76 or a sequence complementary thereto and retains the same binding affinity to said polypeptide of SEQ ID Nos. 1 to
 38. 5. The nucleic acid according to claim 1, encoding a polypeptide having an amino acid sequence consisting essentially of: 45 consecutive amino acids of SEQ ID No. 1; 30 consecutive amino acids of SEQ ID No. 2; 65 consecutive amino acids of SEQ ID No. 3; 30 consecutive amino acids of SEQ ID No. 4; 130 consecutive amino acids of SEQ ID No. 5; 25 consecutive amino acids of SEQ ID No. 6; 23 consecutive amino acids of SEQ ID No. 7; 48 consecutive amino acids of SEQ ID No. 8; 36 consecutive amino acids of SEQ ID No. 9; 25 consecutive amino acids of SEQ ID No. 10; 24 consecutive amino acids of SEQ ID No. 11; 37 consecutive amino acids of SEQ ID No. 12; 25 consecutive amino acids of SEQ ID No. 13; 30 consecutive amino acids of SEQ ID No. 14; 27 consecutive amino acids of SEQ ID No. 15; 69 consecutive amino acids of SEQ ID No. 16; 130 consecutive amino acids of SEQ ID No. 17; 33 consecutive amino acids of SEQ ID No. 18; 25 consecutive amino acids of SEQ ID No. 19; 40 consecutive amino acids of SEQ ID No. 20; 78 consecutive amino acids of SEQ ID No. 21; 39 consecutive amino acids of SEQ ID No. 22; 57 consecutive amino acids of SEQ ID No. 23; 26 consecutive amino acids of SEQ ID No. 24; 68 consecutive amino acids of SEQ ID No. 25; 34 consecutive amino acids of SEQ ID No. 26; 42 consecutive amino acids of SEQ ID No. 27; 48 consecutive amino acids of SEQ ID No. 28; 102 consecutive amino acids of SEQ ID No. 29; 49 consecutive amino acids of SEQ ID No. 30; 92 consecutive amino acids of SEQ ID No. 31; 49 consecutive amino acids of SEQ ID No. 32; 55 consecutive amino acids of SEQ ID No. 33; 69 consecutive amino acids of SEQ ID No. 34; 23 consecutive amino acids of SEQ ID No. 35; 33 consecutive amino acids of SEQ ID No. 36; 32 consecutive amino acids of SEQ ID No. 37; or 22 consecutive amino acids of SEQ ID No.
 38. 6. The nucleic acid according to claim 1 or claim 5, wherein said nucleic acid encodes a polypeptide having one to three amino acid substitutions, wherein said substitutions are made with equivalent amino acids.
 7. A polypeptide consisting essentially of an amino acid sequence of SEQ ID Nos. 1 to 38 or a polypeptide having at least 95% amino acid identity with an amino acid sequence of SEQ ID Nos. 1 to 38 and retains the same binding affinity to said polypeptide of SEQ ID Nos. 1 to
 38. 8. The polypeptide according to claim 7, wherein said polypeptide consists essentially of: 45 consecutive amino acids of SEQ ID No. 1; 30 consecutive amino acids of SEQ ID No. 2; 65 consecutive amino acids of SEQ ID No. 3; 30 consecutive amino acids of SEQ ID No. 4; 130 consecutive amino acids of SEQ ID No. 5; 25 consecutive amino acids of SEQ ID No. 6; 23 consecutive amino acids of SEQ ID No. 7; 48 consecutive amino acids of SEQ ID No. 8; 36 consecutive amino acids of SEQ ID No. 9; 25 consecutive amino acids of SEQ ID No. 10; 24 consecutive amino acids of SEQ ID No. 11; 37 consecutive amino acids of SEQ ID No. 12; 25 consecutive amino acids of SEQ ID No. 13; 30 consecutive amino acids of SEQ ID No. 14; 27 consecutive amino acids of SEQ ID No. 15; 69 consecutive amino acids of SEQ ID No. 16; 130 consecutive amino acids of SEQ ID No. 17; 33 consecutive amino acids of SEQ ID No. 18; 25 consecutive amino acids of SEQ ID No. 19; 40 consecutive amino acids of SEQ ID No. 20; 78 consecutive amino acids of SEQ ID No. 21; 39 consecutive amino acids of SEQ ID No. 22; 57 consecutive amino acids of SEQ ID No. 23; 26 consecutive amino acids of SEQ ID No. 24; 68 consecutive amino acids of SEQ ID No. 25; 34 consecutive amino acids of SEQ ID No. 26; 42 consecutive amino acids of SEQ ID No. 27; 48 consecutive amino acids of SEQ ID No. 28; 102 consecutive amino acids of SEQ ID No. 29; 49 consecutive amino acids of SEQ ID No. 30; 92 consecutive amino acids of SEQ ID No. 31; 49 consecutive amino acids of SEQ ID No. 32; 55 consecutive amino acids of SEQ ID No. 33; 69 consecutive amino acids of SEQ ID No. 34; 23 consecutive amino acids of SEQ ID No. 35; 33 consecutive amino acids of SEQ ID No. 36; 32 consecutive amino acids of SEQ ID No. 37; or 22 consecutive amino acids of SEQ ID No.
 38. 9. The polypeptide according to claim 7, wherein said polypeptide has from one to three, amino acid substitutions, wherein said substitutions are made with equivalent amino acids.
 10. The polypeptide according to claim 8, wherein said polypeptide has from one to three amino acid substitutions, wherein said substitutions are made with equivalent amino acids.
 11. An antibody directed against a polypeptide according to claim 7 or claim
 8. 12. A recombinant vector containing inserted therein a nucleic acid according to claim
 1. 13. A recombinant vector containing inserted therein a nucleic acid according to claim
 2. 14. A recombinant vector containing inserted therein a nucleic acid according to claim
 3. 15. A recombinant vector containing inserted therein a nucleic acid according to claim
 4. 16. A recombinant vector containing inserted therein a nucleic acid according to claim
 5. 17. The recombinant vector according to any one of claims 12 to 16 which is a pACTIIst plasmid or a pAS2ΔΔ plasmid.
 18. The recombinant vector according to any one of claims 12 to 16, which is pT25, pKT25, pUT18 and pUT18C.
 19. The recombinant vector according to any one of claims 12 to 16, which is pP6 and pB5.
 20. A cell host transformed with a vector according to any one of claims 12 to
 16. 21. A set of two nucleic acids consisting essentially of: (i) a first nucleic acid encoding a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to 10; and (ii) a second nucleic acid encoding a prey polypeptide which binds to the SID® polypeptide defined in i).
 22. A set of two nucleic acids consisting essentially of the following: SEQ ID No. 77/SEQ ID No. 1; SEQ ID No. 78/SEQ ID No. 2; SEQ ID No. 78/SEQ ID No. 3; SEQ ID No. 79/SEQ ID No. 4; SEQ ID No. 80/SEQ ID No. 5; SEQ ID No. 81/SEQ ID No. 6; SEQ ID No. 82/SEQ ID No. 7; SEQ ID No. 83/SEQ ID No. 8; SEQ ID No. 84/SEQ ID No. 9; SEQ ID No. 85/SEQ ID No. 10; SEQ ID No. 86/SEQ ID No. 11; SEQ ID No. 87/SEQ ID No. 12; SEQ ID No. 88/SEQ ID No. 13; SEQ ID No. 89/SEQ ID No. 14; SEQ ID No. 90/SEQ ID No. 15; SEQ ID No. 91/SEQ ID No. 16; SEQ ID No. 92/SEQ ID No. 17; SEQ ID No. 93/SEQ ID No. 18; SEQ ID No. 94/SEQ ID No. 19; SEQ ID No. 95/SEQ ID No. 20; SEQ ID No. 96/SEQ ID No. 21; SEQ ID No. 97/SEQ ID No. 22 SEQ ID No. 98/SEQ ID No. 23; SEQ ID No. 99/SEQ ID No. 24; SEQ ID No. 100/SEQ ID No. 25; SEQ ID No. 101/SEQ ID No. 26; SEQ ID No. 102/SEQ ID No. 27; SEQ ID No. 103/SEQ ID No. 28; SEQ ID No. 104/SEQ ID No. 29; SEQ ID No. 105/SEQ ID No. 30; SEQ ID No. 106/SEQ ID No. 31; SEQ ID No. 107/SEQ ID No. 32; SEQ ID No. 108/SEQ ID No. 33; SEQ ID No. 109/SEQ ID No. 34; SEQ ID No. 110/SEQ ID No. 35; SEQ ID No. 111/SEQ ID No. 36; SEQ ID No. 112/SEQ ID No. 37; or SEQ ID No. 113/SEQ ID No.
 38. 23. A set of two polypeptides consisting essentially of: i) a first polypeptide consisting essentially of a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to 10; and ii) a second prey polypeptide which binds specifically with the first polypeptide.
 24. A set of two polypeptides consisting essentially of the following sets: SEQ ID No. 114/SEQ ID No. 39; SEQ ID No. 115/SEQ ID No. 40; SEQ ID No. 115/SEQ ID No. 41; SEQ ID No. 116/SEQ ID No. 42; SEQ ID No. 117/SEQ ID No. 43; SEQ ID No. 118/SEQ ID No. 44; SEQ ID No. 119/SEQ ID No. 45; SEQ ID No. 120/SEQ ID No. 46; SEQ ID No. 121/SEQ ID No. 47; SEQ ID No. 122/SEQ ID No. 48; SEQ ID No. 123/SEQ ID No. 49; SEQ ID No. 124/SEQ ID No. 50; SEQ ID No. 125/SEQ ID No. 51; No. 126/SEQ ID No. 52; SEQ ID No. 127/SEQ ID No. 53; SEQ ID No. 128/SEQ ID No. 54; SEQ ID No. 129/SEQ ID No. 55; SEQ ID No. 130/SEQ ID No. 56; SEQ ID No. 131/SEQ ID No. 57; SEQ ID No. 132/SEQ ID No. 58; SEQ ID No. 133/SEQ ID No. 59; SEQ ID No. 134/SEQ ID No. 60; SEQ ID No. 135/SEQ ID No. 61; SEQ ID No. 136/SEQ ID No. 62; SEQ ID No. 137/SEQ ID No. 63; SEQ ID No. 138/SEQ ID No. 64; No. 139/SEQ ID No. 65; SEQ ID No. 140/SEQ ID No. 66; SEQ ID No. 141/SEQ ID No. 67; SEQ ID No. 142/SEQ ID No. 68; SEQ ID No. 143/SEQ ID No. 69; SEQ ID No. 144/SEQ ID No. 70; SEQ ID No. 145/SEQ ID No. 71; SEQ ID No. 146/SEQ ID No. 72; SEQ ID No. 147/SEQ ID No. 73; SEQ ID No. 148/SEQ ID No. 74; SEQ ID No. 149SEQ ID No. 75; or SEQ ID No. 150/SEQ ID No.
 76. 25. A complex formed between said set of two polypeptides of claim
 23. 26. A complex formed between said set of two polypeptides of claim
 24. 27. A method for selecting a molecule which inhibits the binding between a set of two polypeptides wherein said method comprises: a) cultivating a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two vectors wherein: i) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said two polypeptides according to claim 23 and a DNA binding domain; ii) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides according to claim 23 and an activating domain capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting; on a selective medium containing the molecule to be tested and allowing the growth of said recombinant host cell when the toxic reporter gene is not activated; and b) selecting the molecule which inhibits the growth of the recombinant host cell defined in step a).
 28. A method for selecting a molecule which inhibits the binding between a set of two polypeptides wherein said method comprises: c) cultivating a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two vectors wherein: iii) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said two polypeptides according to claim 24 and a DNA binding domain; iv) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides according to claim 24 and an activating domain capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting; on a selective medium containing the molecule to be tested and allowing the growth of said recombinant host cell when the toxic reporter gene is not activated; and b) selecting the molecule which inhibits the growth of the recombinant host cell defined in step a).
 29. A method for selecting a molecule which inhibits protein-protein interaction of a set of two polypeptides, wherein said method comprises the steps of: a.) cultivating a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two vectors wherein: i) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said set of two polypeptides according to claim 23 and the first domain of an enzyme; ii) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides according to claim 23 and the second part of said enzyme capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting, said interaction recovering the catalytic activity of the enzyme; on a selective medium containing the molecule to be tested and allowing the growth of said recombinant host cell when the toxic gene is not activated; and d) selecting the molecule which inhibits the growth of the recombinant host cell defined in step a).
 30. A method for selecting a molecule which inhibits protein-protein interaction of a set of two polypeptides, wherein said method comprises the steps of: a) cultivating a recombinant host cell containing a reporter gene the expression of which is toxic for said recombinant host cell, said host cell being transformed with two vectors wherein: i) the first vector contains a nucleic acid comprising a polynucleotide encoding a first hybrid polypeptide containing one of said set of two polypeptides according to claim 24 and the first domain of an enzyme; ii) the second vector contains a nucleic acid comprising a polynucleotide encoding a second hybrid polypeptide containing the second of said two polypeptides according to claim 24 and the second part of said enzyme capable of activating said toxic reporter gene when the first and the second hybrid polypeptides are interacting, said interaction recovering the catalytic activity of the enzyme; on a selective medium containing the molecule to be tested and allowing the growth of said recombinant host cell when the toxic gene is not activated; and b) selecting the molecule which inhibits the growth of the recombinant host cell defined in step a).
 31. A marker compound wherein said marker compound comprises: a.) a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to 10; and b.) a detectable molecule bound thereto.
 32. The marker compound of claim 31, wherein the detectable molecule is a fluorescent protein.
 33. The marker compound of claim 32, wherein the detectable protein is a green fluorescent protein or a yellow flourescent protein.
 34. The marker compound of claim 31, wherein the detectable molecule has a catalytic activity.
 35. The marker compound of claim 34, wherein the detectable molecule is a hydrolase, a transferase, a lyase, an isomerase, a ligase, a synthetase or a oxidoreductase.
 36. The marker compound of claim 31, wherein the detectable molecule is a radioactive molecule.
 37. The marker compound of claim 31, wherein the detectable molecule is chemiluminescent molecule.
 38. The marker compound of claim 31, wherein the detectable molecule is covalently bound to said Selected Interacting Domain (SID®) polypeptide.
 39. The marker compound of claim 31, wherein the detectable molecule is non covalently bound to the Selected Interacting Domain (SID®) polypeptide.
 40. The marker compound of claim 31, wherein the detectable molecule is an antibody directed against the Selected Interacting Domain(SID®)polypeptide.
 41. The marker compound of claim 40, wherein said antibody is labeled radioactively or nonradioactively.
 42. The marker compound according to claim 31, wherein: a.) said Selected Interacting Domain (SID®) polypeptide is covalently bound to a first ligand; and b.) said detectable molecule comprises a second ligand which binds specifically to the first ligand.
 43. The marker compound according to claim 42, wherein the first ligand is biotin and the second ligand is streptavidin.
 44. A nucleic acid encoding a marker compound according to claim
 42. 45. A recombinant vector comprising inserted therein a nucleic acid according to claim 44
 46. The recombinant vector according to claim 45, which is pACTIIst, pASΔΔ, pT25, pKT25, pUT18, pUT18C, pP6 or pB5.
 47. A recombinant host cell which has been transfected with said recombinant vector according to claim
 45. 48. The recombinant host cell according to claim 45 which is of prokaryotic origin.
 49. The recombinant host cell according to claim 45 which is of eukaryotic origin.
 50. The recombinant host cell according to claim 49 which is a mammalian host cell.
 51. A method of detecting a polypeptide of interest in a sample, said method comprising: a) contacting a marker compound or a plurality of marker compounds according to claim 31 with said sample; and b) detecting the complexes formed between said marker compound or plurality of marker compounds and said polypeptide of interest.
 52. The method according to claim 51, wherein said marker compound is inserted in a recombinant vector.
 53. The method according to claim 51, wherein said marker compound is in a recombinant host cell.
 54. A method for detecting a polypeptide or a plurality of polypeptides of interest in a sample, wherein said method comprises: a) providing a substrate onto which a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to 10, or a pluralality of Selected Interacting Domain (SID®) polypeptides according to any one of claims 7 to 10 is immobilized; b) bringing into contact the substrate defined in a) with the sample to be assayed; c) detecting the complexes formed between the Selected Interacting Domain (SID®) polypeptides or variants thereof or a variant thereof, or the plurality of Selected Interacting Domain (SID®) polypeptides and a molecule or a plurality of molecules initially contained in the sample.
 55. The method of claim 54, wherein the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides are covalently bound to the substrate.
 56. The method of claim 54, wherein the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides or variants thereof are non covalently bound to the substrate.
 57. The method if claim 54, wherein the Selected Interacting Domain (SID®) polypeptide or the plurality of Selected Interacting Domain (SID®) polypeptides are covalently bound to a first ligand and wherein the substrate is coated with a second ligand which specifically binds to the first ligand.
 58. The method of claim 57, wherein the first ligand is biotin and the second ligand is streptavidin.
 59. The method according to claim 54, wherein the Selected Interacting Domain (SID®) polypeptide or a variant thereof, or the plurality of Selected Interacting Domain (SID®) polypeptides or variants thereof are covalently linked to a spacer and wherein said spacer is covalently bound to the substrate in order to immobilize the Selected Interacting Domain (SID®) polypeptide or a variant thereof, or the plurality of Selected Interacting Domain (SID®) polypeptides.
 60. The method according to claim 54, wherein the detection step c) consists of detecting changes in the optical characteristics of the substrate.
 61. A device for the detection of a polypeptide or a plurality of polypeptides of interest within a sample, said device comprises a substrate onto which a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to 10 or a plurality thereof is immobilized.
 62. A pharmaceutical composition comprising a pharmaceutically effective amount of a nucleic acid comprising a polynucleotide encoding a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to
 10. 63. A method for treating a viral infection by a Hepatitis C virus in a human or animal in need of such treatment, said method comprising administering to said human or said animal a pharmaceutically effective amount of a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to
 10. 64. A method for treating a viral or a bacterial infection in a human or an animal in need of such treatment, said method comprising administering to said human or said animal a pharmaceutically effective amount of a recombinant expression vector comprising a polynucleotide encoding a Selected Interacting Domain (SID®) polypeptide according to any one of claims 7 to
 10. 65. A method for selecting a Selected Interacting Domain (SID®) polypeptide comprising: 1) selecting a collection of nucleic acids (prey nucleic acids) which bind specifically to a given bait polypeptide of interest; and 2) determining the nucleic acid sequences which encode for a SID® polypeptide by: a) selecting from the collection of prey nucleic acids obtained at the end of step 1) all prey nucleotides encoding a prey polypeptide capable of interacting with said bait polypeptide and containing a common nucleic acid fragment; b) aligning the nucleotide sequences of the prey polynucleotides as selected at step a) and gathering in one set or in a plurality of sets of sequences those nucleotide sequences which have sequences that overlap for more than 30% of their respective nucleic acid length, wherein each common overlapping nucleotide sequence in one set of sequences defines a sequence encoding a pre-SID® polypeptide; and c) aligning two sequences encoding two respective pre-SID® polypeptides; and: i) defining an overlapping nucleic acid sequence between the sequences encoding the two respective pre-SID® polypeptides as a sequence encoding a SID® polypeptide, provided that the overlapping sequence is of at least 30 nucleotides in length; ii) defining a non-overlapping nucleic acid sequence between the sequences encoding the two respective pre-SID® polypeptides as a sequence encoding a SID® polypeptide, provided that (1) said non-overlapping sequence has more than 30 nucleotides in length and (2) said non-overlapping sequence represents at least 30% in length of any one of the polynucleotides contained in the set of prey polynucleotides used for defining the sequence encoding each pre-SID® polypeptide.
 66. The method of claim 65, wherein said selection step 1) uses a yeast two-hybrid method or a bacterial two-hybrid method.
 67. The method of claim 65, wherein step 2) further comprises the steps of: d) counting the number of overlapping prey polynucleotides contained in a first set of polynucleotides defining a sequence encoding a first SID® polypeptide; e) counting the number of overlapping prey polynucleotides contained in a second set of polynucleotides defining a sequence encoding a second SID® polypeptide which overlaps with the sequence encoding the first second SID® polypeptide; f) determining which sequence among those encoding respectively the first SID® polypeptide and the second SID® polypeptide has been defined with the largest number of prey polynucleotides and selecting this set of prey sequences; g) adding to the set of prey sequences selected at step f) those sequences that were contained in the set of prey sequences used for defining the sequence encoding the SID® polypeptide with the smallest number of prey sequences and which overlap with the sequence encoding the SID® polypeptide with the largest number of prey sequences; h) aligning the prey sequences added at step g) with the sequences already contained in the set of prey sequences which defined the sequence encoding the SID® polypeptide with the largest number of prey sequences; and i) defining an overlapping sequence between the whole sequences which were aligned in step h), wherein said overlapping sequence consists of a sequence encoding a SID® polypeptide.
 68. The method according to claim 65, wherein said SID® is from a virus.
 69. The method according to claim 68, wherein the virus is a Hepatitis C virus.
 70. The method according to claim 69, wherein the Hepatitis C virus is pathogenic for a mammal.
 71. The method according to claim 70, wherein said mammal is a human.
 72. A SID® nucleic acid selected according to the method of claim
 65. 73. A SID® polypeptide encoded by nucleic acid according to claim
 72. 